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Chem 333 – Metabolism

Lecture Notes

Michael Palmer
Department of Chemistry
University of Waterloo
Waterloo, Ontario, Canada

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Preface

These notes are for use with my 3rd year undergraduate class on metabolism.
The focus is on human metabolism, and more specifically on human energy me-
tabolism. Apart from the pathways and reactions, several aspects of hormonal
regulation and some medical correlations are also included. The entire text
has been prepared by myself. The same applies to most figures; exceptions are
indicated. With respect to my own material, you are welcome to use it in your
own not-for-profit teaching materials as you see fit, provided that proper credit
is given.
These notes are currently in their 4th edition. In this edition, some errors
have been corrected, yet likely some more remain to be discovered, and new
ones may have been introduced. I therefore welcome corrections and sug-
gestions for improvement, no matter whether you’re a colleague, a student,
or simply an interested reader. Please send email to mpalmer@uwaterloo.ca.
Thank you.
Contents

Chapter 1 Introduction 1
1.1 Motivation: Why would you study metabolism? . . . . . . . . . . 1
1.2 Catabolic and anabolic reactions . . . . . . . . . . . . . . . . . . . 1
1.3 Metabolic diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Types of foodstuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 The digestive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.6 What’s next? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Chapter 2 Refresher 16
2.1 How enzymes work: Active sites and catalytic mechanisms . . 16
2.2 Classification of Enzymes and enzyme reactions . . . . . . . . . 18
2.3 Energetics of enzyme-catalyzed reactions . . . . . . . . . . . . . 19
2.4 The role of ATP in enzyme-catalyzed reactions . . . . . . . . . . 21
2.5 Regulation of enzyme activity . . . . . . . . . . . . . . . . . . . . 23

Chapter 3 Glycolysis 26
3.1 Overview of glucose metabolism . . . . . . . . . . . . . . . . . . . 26
3.2 The place of glycolysis in glucose degradation . . . . . . . . . . 27
3.3 Reactions in glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.4 Mechanisms of enzyme catalysis in glycolysis . . . . . . . . . . 28
3.5 Energy-rich functional groups in substrates of glycolysis . . . . 35
3.6 Function of glycolysis under anaerobic conditions . . . . . . . . 36
3.7 Transport and utilization of glucose . . . . . . . . . . . . . . . . 37

Chapter 4 Catabolism of sugars other than glucose 40


4.1 Metabolism of sucrose and fructose . . . . . . . . . . . . . . . . . 41
4.2 Metabolism of lactose and galactose . . . . . . . . . . . . . . . . 42
4.3 The polyol pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

iii
iv CONTENTS

Chapter 5 Pyruvate dehydrogenase and the citric acid cycle 48


5.1 Pyruvate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . 48
5.2 PDH: Catalytic mechanisms . . . . . . . . . . . . . . . . . . . . . . 52
5.3 Regulation of pyruvate dehydrogenase . . . . . . . . . . . . . . . 52
5.4 The citric acid cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.5 Reactions in the citric acid cycle . . . . . . . . . . . . . . . . . . . 56
5.6 Regulation of the citric acid cycle . . . . . . . . . . . . . . . . . . 59

Chapter 6 The respiratory chain 60


6.1 ATP synthesis can be separated from electron transport . . . . 62
6.2 The electron transport chain . . . . . . . . . . . . . . . . . . . . . 65
6.3 ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.4 The ATP yield of oxidative glucose degradation . . . . . . . . . 79
6.5 Shuttle systems for the NADH re-oxidation . . . . . . . . . . . . 80
6.6 Regulation of the respiratory chain . . . . . . . . . . . . . . . . . 82

Chapter 7 Gluconeogenesis 85
7.1 Reactions in gluconeogenesis . . . . . . . . . . . . . . . . . . . . . 86
7.2 Glucogenic amino acids and gluconeogenesis . . . . . . . . . . . 88
7.3 Regulation of gluconeogenesis . . . . . . . . . . . . . . . . . . . . 88
7.4 Energy balance of gluconeogenesis . . . . . . . . . . . . . . . . . 92
7.5 The Cori cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.6 The glyoxylate cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
7.7 Additional roles of gluconeogenetic enzymes . . . . . . . . . . . 94

Chapter 8 Glycogen metabolism 97


8.1 Glycogen synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
8.2 Glycogen degradation . . . . . . . . . . . . . . . . . . . . . . . . . . 101
8.3 Glycogen storage diseases . . . . . . . . . . . . . . . . . . . . . . . 101
8.4 Regulation of glycogen metabolism . . . . . . . . . . . . . . . . . 103

Chapter 9 The hexose monophosphate shunt 105


9.1 Reactions in the hexose monophosphate shunt . . . . . . . . . . 106
9.2 Mechanisms of transketolase and transaldolase . . . . . . . . . 110
9.3 Why do we need both NADH and NADPH? . . . . . . . . . . . . . 111
9.4 Alternative sources of NADPH . . . . . . . . . . . . . . . . . . . . 111
9.5 Uses of NADPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
9.6 Glucose-6-phosphate dehydrogenase deficiency . . . . . . . . . 115
CONTENTS v

Chapter 10 Triacylglycerol metabolism 118


10.1 Utilization of dietary triacylglycerol . . . . . . . . . . . . . . . . . 120
10.2 Utilization of fatty acids: β-Oxidation . . . . . . . . . . . . . . . . 124
10.3 Triacylglycerol utilization . . . . . . . . . . . . . . . . . . . . . . . 128
10.4 Ketone bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
10.5 Fatty acid synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
10.6 Pharmacological inhibition of fatty acid synthase . . . . . . . . 139

Chapter 11 Cholesterol metabolism 141


11.1 Uptake and transport of cholesterol . . . . . . . . . . . . . . . . . 142
11.2 Distribution of cholesterol from the liver to the periphery . . . 142
11.3 Transport of excess cholesterol to the liver . . . . . . . . . . . . 143
11.4 Esterification of cholesterol . . . . . . . . . . . . . . . . . . . . . . 144
11.5 Synthesis of cholesterol . . . . . . . . . . . . . . . . . . . . . . . . 144
11.6 The endoplasmic reticulum in steroid synthesis . . . . . . . . . 146
11.7 7-Dehydrocholesterol and vitamin D3 . . . . . . . . . . . . . . . . 148
11.8 Regulation of cholesterol synthesis . . . . . . . . . . . . . . . . . 149
11.9 Cholesterol in atherosclerosis . . . . . . . . . . . . . . . . . . . . 151
11.10 Therapy of hypercholesterolemia . . . . . . . . . . . . . . . . . . 152

Chapter 12 Amino acid metabolism 154


12.1 Overview of amino acid degradation . . . . . . . . . . . . . . . . 155
12.2 Transamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
12.3 The urea cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
12.4 Auxiliary reactions in nitrogen transport and elimination . . . 161
12.5 Degradative pathways of individual amino acids . . . . . . . . . 165
12.6 Hereditary enzyme defects in amino acid metabolism . . . . . 170

Chapter 13 Hormonal regulation of metabolism 176


13.1 Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
13.2 Glucagon and epinephrine . . . . . . . . . . . . . . . . . . . . . . . 184
13.3 Glucocorticoids and thyroid hormones . . . . . . . . . . . . . . . 186
13.4 Leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Chapter 14 Diabetes mellitus 189


14.1 The role of insulin and the causation of diabetes . . . . . . . . . 190
14.2 Effects of insulin deficiency on carbohydrate metabolism . . . 190
14.3 Insulin deficiency and lipid metabolism . . . . . . . . . . . . . . 192
14.4 Laboratory findings and clinical symptoms in acute type I
diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
14.5 The cause of β-cell destruction in type I diabetes . . . . . . . . 194
14.6 Why do diabetic patients lose glucose in the urine? . . . . . . . 194
14.7 Treatment of type I diabetes . . . . . . . . . . . . . . . . . . . . . 196
14.8 Long-term complications of diabetes mellitus . . . . . . . . . . . 197
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14.9 Glucose assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199


14.10 Diabetes type II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
14.11 Diabetic coma and comatose diabetics . . . . . . . . . . . . . . . 201
14.12 Other forms of diabetes . . . . . . . . . . . . . . . . . . . . . . . . 202
14.13 The End . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Chapter 1

Introduction

1.1 Motivation: Why would you study metabolism?


Long answer: You have a generous and warm character, and you have spent
too much time in front of the family TV set. You are therefore determined
to become a famous doctor and save many, many lives every hour of the day,
without asking anything in compensation but the admiring gaze of the populace,
and may be a Rolls Royce. Metabolism is an ever so tiny part of the vast
knowledge you have set out to master in order to fulfill your destiny.
Wrong? Try this one: You have the inquisitive mind of a Sherlock Holmes
and the financial savvy of a Howard Hughes, and you have determined that
soaking medical doctors for damages is the best road to wealth and fame.
Understanding the biochemical basis of medicine will help you to stun your
audiences in court and grind the defendants and their counsels into the dust.
Wrong again? Then try the short answer: You want to pass your exam.

1.2 Catabolic and anabolic reactions


Metabolism is a central theme in biochemistry. Metabolism keeps cells and
organisms alive, by giving them the energy to carry on and the building blocks
required for growth and propagation. The metabolism of animals and humans,
which depend entirely on foodstuffs, can be divided into catabolic and anabolic
reactions.
The term catabolic means the same as degradative, but it is Greek and
therefore sounds a whole lot more erudite and scholarly. Key functions of
catabolic reactions are

1
2 1 Introduction

Foodstuffs small intermediates

complex
biomolecules ADP+Pi
NADP+ O2

NADPH+H+
ATP
small intermediates CO2 + H2O

Figure 1.1 A broad outline of human metabolism. Foodstuffs, which are mostly ma-
cromolecules, are broken down in catabolic reactions to small intermediates, which in
turn are in part further degraded to yield energy (ATP) and reducing power (NADPH).
Some of the intermediates thus produced are used in anabolic reactions for the synthe-
sis of new macromolecules. These may in turn be recycled and used as substrates for
the release of small intermediates.

1. accumulation of energy in the form of ATP,


2. regeneration of reducing power (NADPH), and
3. production of building blocks for anabolic metabolism.
A large share of the substrates broken down in catabolism are being used
for producing ATP, the “electric energy” of the cell. Just as electricity can
be used to drive just about any household job, ATP is used for nearly every
energy-requiring task in cell biology. This includes
1. cell motility, particularly in muscle cells,
2. active transport across membranes, e.g. Na+/K+-ATPase and other ion
pumps. Ion pumps are largely responsible for the conspicuous energy
requirements of brain and kidneys,
3. anabolic metabolism.
Because of its key role in the life of the cell, we will devote a good deal of time
to the metabolic pathways that allow the cell to regenerate ATP.
Anabolic reactions are the opposite of catabolic ones, that is they create new
biomolecules. These include small molecules and building blocks that are not
sufficiently available in the food, and macromolecules, in particular proteins
and nucleic acids. Apart from building blocks and ATP, anabolic reactions
also require a good deal of reducing power in the form of NADPH, and we will
accordingly look at the major pathway devoted to its production, which is the
hexose monophosphate shunt (chapter 9).
1.2 Catabolic and anabolic reactions 3

UDP-Galactose UDP-Glucose Glycogen

Glucose 1-P 6-P-Gluconate Ribulose 5-P


Galactose Galactose 1-P

Lactose Glucose Glucose 6-P

Amylose Fructose 6-P Erythrose 4-P


Sucrose Xylulose 5-P
Sedoheptulose 7-P
Fructose Fructose 1,6-bis-P

Ribose 5-P
Fructose 1-P Glyceraldehyde Glyceraldehyde 3-P/
Dihydroxyacetone-P

1,3-bis-P-glycerate
Glycerol-P Glycerol
3-P-glycerate
Triacylglycerol
2-P-glycerate

Phosphoenolpyruvate (PEP) Fatty acyl-CoA Fatty acids


Ala
Cys
Pyruvate
Ser
Malonyl-CoA Gly
CO2
Acetyl-CoA HMG-CoA Acetoacetate Leu
CO2 NH3 Lys
β-Hydroxybutyrate Phe
Carbamoyl-P Trp
Asp Oxaloacetate Citrate
Citrulline Tyr
Cholesterol
Malate Isocitrate Gln
H2 CO2 Arg
Ornithine Argininosuccinate
Fumarate α-Ketoglutarate Glu His
Pro
CO2
Urea Arginine Succinate Succinyl-CoA
Methylmalonyl-CoA
CO2
Phe Ile
Tyr O2 ADP + Pi Propionyl-CoA Met
Thr
ATP Val
Odd-numbered
H2O fatty acids

Figure 1.2 Metabolic pathways covered in this class. Solid arrows indicate single
enzyme reactions, with the exception of the respiratory chain, which converts H2 to
H2 O. Dashed arrows represent sequences of enzyme reactions.
4 1 Introduction

The relationships described above between catabolic and anabolic pathways


are summarized in Figure 1.1.

1.3 Metabolic diversity


There are several mainstream metabolic pathways that occur in a wide variety
of living organisms. A good example is glycolysis, the main pathway of glucose
degradation. On the other hand, it is important to note that metabolic processes
in organisms other than man or animals may be quite different. Examples are:
1. Photosynthesis, which ultimately enables plants to create all their carbon
compounds from CO2 and water. However, even plants do perform catabolic
reactions on endogenous macromolecules. An example is the degradation of
starch, which is stored in large amounts in plant seeds (e.g. wheat and rice) and
bulbs (e.g. potatoes). The pathways of starch utilization employed by plants
are entirely analogous to those found in animals.
2. Nitrogen fixation by the bacterium Rhizobium meliloti and related species.
Most organisms require nitrogen in reduced form (as ammonia or as part of
amino acids), but Rhizobium is able to convert athmospheric nitrogen (N2 ) to
ammonia. This is a process of fundamental importance in agriculture (and in
fact, to life as such), since the ammonia is required by plants for amino acid
synthesis.
3. Some bacteria that live in quite exotic environments have developed corre-
spondingly exotic metabolic pathways. Some of these are capable of extracting
energy from the oxidation of iron or the reduction of sulfur.
While these pathways are certainly very interesting, we will not deal with
them in this class. Instead, we will look at only those metabolic pathways that
occur in human cells, and not even at all of these. The focus will be on pathways
that supply the cell with ATP and reducing power for its various tasks, that
is on catabolic pathways. A summary of the pathways covered in this class is
given in Figure 1.2. In addition, we will relate some of these pathways to human
health and disease.

1.4 Types of foodstuffs


Catabolism starts with foodstuffs. The three major categories of foodstuffs
relevant to human metabolism are named on every box of cereal or cup of
yoghurt (Figure 1.3a). If you look at the top of Figure 1.2, you will see lots of
names ending in -ose. These are sugars. Glucose, fructose, and galactose are
single sugar molecules, or monosaccharides. Sucrose und lactose are dimeric
sugars or disaccharides, whereas amylose is polymeric, or a polysaccharide.
Utilization of disaccharides and oligosaccharides starts with their cleavage to
monosaccharides.
1.5 The digestive tract 5

The sum formulas of sugars can approximately be written as (CH2 O)n , that
is they formally are multiples of carbon (C) plus water (H2 O). They are therefore
collectively referred to as carbohydrates. These form an important part of our
diet. Indeed, in the typical diets of many countries, the most calories by far are
derived from carbohydrates, contained for example in rice, wheat, or potatoes.
To the right of the center in Figure 1.2 you will find triacylglycerol. This is
another major component in our diet: Fat. The decomposition of fat yields fatty
acids as the main component; these are further degraded in the β-oxidation
pathway to acetyl-CoA. Acetyl-CoA is also an intermediate in the complete
degradation of carbohydrates and of proteins. Therefore, acetyl-CoA is a cen-
tral hub in metabolism, through which all substrates destined for complete
oxidative degradation must pass. Acetyl-CoA is also a precursor in several
biosynthetic pathways (Figure 1.3b).
The third major type of foodstuff are proteins. Of the twenty standard
amino acids found in proteins, we can synthesize only ten ourselves; the other
ones are called essential amino acids. Therefore, while a low amount of dietary
carbohydrates or fat can be compensated by our own biosynthesis,1 lack of
dietary protein cannot. Accorgingly, in poor countries, lack of dietary protein
is the most common form of malnutrition.
One class of macromolecules that is not covered here are the nucleic acids.
Nevertheless, nucleic acids make up a sizeable fraction of our food. Only the
sugar part of each nucleotide—ribose or deoxyribose—can be utilized to gain
energy. The bases—A,C,G,T—can be used as building blocks for the synthe-
sis of new nucleotides and nucleic acids. If they are present in excess over
biosynthetic demands, they are modified and excreted.

1.5 The digestive tract

Where does metabolism occur? The first step, the depolymerization of food-
stuff macromolecules, occurs extracellularly, certainly in humans but typically
even with organisms as simple as bacteria. Depolymerization is accomplished
by digestive enzymes, which are secreted by the cell, and uptake of substrates
into the cell only occurs at the stage of the monomeric breakdown products.
Why is that so? Extracellular digestion makes the substrates available to ev-
eryone, not just to the cell that has provided the necessary enzymes. It is
therefore a potentially ineffective process, especially with unicellular organ-
isms that often will find themselves competing for substrates with numerous
other species.

1 The traditional diet of eskimos, for example, is extremely low in carbohydrates, since it

consists mostly of meat and fish.


6 1 Introduction

a) Foodstuffs

Carbohydrates Sugars CO2 + H2O

Fatty acids,
Fat CO2 + H2O
glycerol

Protein Amino acids CO2 + H2O

Urea, sulfate

b) Sugars Fat Amino acids

ADP ATP
Acetyl-CoA CO + H O
2 2

Fat Cholesterol Ketone bodies


Figure 1.3 Summary of catabolism of the three major classes of foodstuffs. a: All
three types can be completely broken down to carbon dioxide and water to yield ATP
and / or NADPH (see Figure 1.1, right). Amino acids, which contain nitrogen and in
some cases sulfur, also yield urea and sulfate when completely degraded. b: Acetyl-
CoA is a central intermediate in the breakdown of all classes of foodstuffs. It is also a
precursor in the synthesis of fat, cholesterol, and ketone bodies.

An obvious answer is that there are no transport mechanisms for the uptake
of macromolules across the cell wall. While that is true,2 there is a deeper
reason – taking up macromolecules in a non-specific way would open the door
for all kinds of Trojan horses. Extracellular digestion is a kind of a firewall
to exclude hazardous biomolecules.3 Extracellular depolymerization is also
the strategy employed by our own digestive tract (Figure 1.4). The digestive
2 But not universally – for example, bacterial cells have evolved mechanisms for the active

uptake of DNA, which they switch on under special circumstances. This is exploited in the lab in
transformation experiments.
3 Amoebae, for example, do indeed ingest not only macromolecules but even whole bacteria.

They can do so because they confine the ingested material within phagosomes, which they swiftly
flood with acid and aggressive chemicals and enzymes to kill and degrade the bacteria. The same
occurs in our phagocytes, which are an essential part of our immune system.
1.5 The digestive tract 7

a) b)

stomach

liver
liver
pancreas

bile
stomach
bladder

small large bile


intestine intestine duct

pancreas

pancreatic duct
duodenum

Figure 1.4 Schematic of the digestive tract. a: Overview. b: The liver and the pancreas
both have secretory ducts that discharge into the duodenum, which is the topmost part
of the small intestine.

tract contains specialized organs and cells for the secretion of depolymerizing
enzymes, for the performance of the digestion, and finally the uptake of the
released substrates.

1.5.1 The stomach


The first major section of the digestive tract is the stomach. The stomach
contains gastric acid, which is hydrochloric acid (HCl) with a pH of ~2, which
of course creates a very aggressive environment. The gastric acid has two
important biological effects:
1. It sterilizes the food, that is it kills most of the ingested microorganisms.
Individuals with impaired secretion of gastric acid or those who take drugs
that inhibit acid secretion, which is necessary in the treatment of gastric or
duodenal ulcers, are more susceptible to infectious diseases such as cholera
and intestinal tuberculosis.
2. It denatures (or unfolds) proteins. In denatured form, proteins are much
more accessible to digestion. Protein digestion is initiated right away in the
stomach by the protease pepsin. The peptides generated will no longer refold,
even though the pH returns to near neutral conditions in the small intestine,
so that digestion can be completed by the proteases and peptidases found
there (Figure 1.5). Most, but not all proteins will be unfolded by gastric acid;
an important exception is pepsin itself. The coat proteins of many pathogenic
viruses, for example poliovirus, are fairly resistant to gastric acid as well, so
8 1 Introduction

Pepsin

pH 2

Trypsin etc.

pH 7.5

Uptake of small peptides


and free amino acids

Figure 1.5 Digestion of proteins. The acidic pH in the stomach causes the proteins
to unfold, which makes the peptide bonds accessible to pepsin. The fragments will not
be able to refold, even though the pH reverts to neutral in the small intestine, so that
the digestion can be completed, and the final products be taken up.

that these viruses are able to traverse the stomach and then infect the intestinal
mucosa.

1.5.2 The pancreas


While the stomach protects the lower parts of the intestinal tract from infec-
tious agents and conditions the food for further digestion, most of the hard
work is done in the small intestine. For the digestion to occur, we need specific
enzymes for each of the major polymers that occur in the food:
1. Amylases and disaccharidases degrade starch (Figure 1.6) and other car-
bohydrates.
2. Proteases and peptidases degrade proteins and release amino acids.
3. Lipases digest fat to release glycerol and fatty acids.
4. Nucleases release nucleotides from nucleic acids.
Most of these digestive enzymes are synthesized by the pancreas. This is
a large gland situated right next to the topmost part of the small intestine,
the duodenum, into which it discharges its secretions. Accordingly, patients
with a lack of pancreas function are deficient in the digestion of all kinds of
foodstuffs.
Apart from the degradative enzymes, the pancreatic juice contains sodium
bicarbonate. The bicarbonate serves to promptly neutralize the gastric acid
upon entering into the duodenum. In keeping with the near-neutral pH that
prevails in the intestine, all the pancreatic enzymes have a roughly neutral pH
optimum, which contrasts with the acidic pH optimum of pepsin.
1.5 The digestive tract 9

O
HC
H OH
CH 2OH CH 2OH
OH
O HO H O
OH OH
H OH
OH OH OH
OH H OH OH

H OH
H

α-D-Glucose D-Glucose β-D-Glucose

CH 2OH CH 2OH CH 2OH CH 2OH

… HO
OH
O

O
OH
O

O
OH
O

O
OH
O

O
OH OH OH
H CH 2OH
OH branch
CH 2OH CH 2OH CH 2OH CH 2
O O O O O O
OH OH OH OH OH
O O O O O O
OH OH OH OH OH OH

Amylopectin

CH 2OH CH 2OH
O O
OH OH
OH O OH
OH OH

Maltose
Figure 1.6 Structures of glucose, amylose and maltose. Top: Glucose occurs in two
anomeric ring forms, as well in an open chain form. Middle: Amylopectin is a branched
polymer of α-glucose. The unbranched polymer is called amylose; both are contained
in starch. Bottom: Maltose is the disaccharide which results from the degradation of
amylose by amylase. It is in turn cleaved to glucose by maltase at the surface of the
intestinal mucosa.

1.5.3 The liver

Among its many other functions, the liver also serves as an exocrine gland.
The digestive juice secreted by the liver is known as the bile and is rich in bile
acids (see Figure 11.1), which are important in solubilizing fat so as to render it
10 1 Introduction

accessible to enzymatic cleavage.4 The bile may be stored and concentrated in


the gall bladder, or directly secreted together with the pancreatic juice (the bile
duct and the pancreatic duct join immediately before reaching the duodenum).
Like the pancreatic juice, the bile is rich in sodium bicarbonate.
In keeping with the occurrence in the bile of bile acids but not of digestive
enzymes, patients suffering from a disruption of bile secretion will show a
deficiency in the digestion of fat but not of proteins or carbohydrates. Apart
from the secretion of bile, the liver has a key role in processing substrates after
uptake from the intestine. This is discussed in section 1.5.6.

1.5.4 The small intestine

After digestion, the metabolites have to be taken up by the cells at the sur-
face of the mucosa of the small intestine, which comprises the duodenum,
the jejunum, and the ileumsmall intestinesections of. How does this uptake
work? Most nutrients are taken up by active transport, which can transport
solutes energetically uphill, that is against their concentration gradients. Active
transport is necessary for the quantitative uptake of the nutrients. An impor-
tant example is the transport of glucose, the most important end product of
carbohydrate digestion. The uptake of glucose is driven by the simultaneous
uptake of two sodium ions per molecule of glucose; this coupling is effected
by the SGLT1 transporter (Figure 1.7). While sodium is plentiful in the gut
lumen, its concentration is low inside the cells. The uphill transport of glucose
is therefore driven by the simultaneous downhill movement of sodium. Similar
transporters exist for other sugars (e.g. galactose) and for amino acids.
The amount of nutrient substrates that have to pass across the surface of
the gut is considerable; therefore, a large active surface is required. You have
probably heard that the length of the small intestine is likewise considerable –
about twice the height of your body. In addition, the inner surface of the small
intestine is folded, and the folds in turn have a shaggy surface, as have the
individual cells. All these factors combine to maximize the effective surface
available for substrate uptake. This is illustrated in Figure 1.8.

1.5.5 The lower small intestine and the large intestine

All digestion and nutrient absorption occurs in the small intestine, mostly in its
upper portions. In fact, even the lower segments of the small intestine usually
don’t get to do much work in nutrient uptake but have a more specialized role
in the reuptake of bile acids, and of things such as vitamin B12 , the lack of
which causes pernicious anemia. In the large intestine, essentially no nutrients
are left for absorption. What, then, is the function of the large intestine? A
4 Bile acids solubilize fat because they are detergents, and as such they are also useful for

removing tough stains from your laundry.


1.5 The digestive tract 11

gut lumen cytosol blood

2 Na+ 2 Na+

Glucose Glucose Glucose

SGLT1 GLUT2

Figure 1.7 Mechanism of glucose uptake from the gut lumen. The transport against
the concentration gradient is powered by cotransport of sodium ions via the trans-
porter SGLT1 (SGLT=sodium glucose transporter), while the transport into the blood is
down a concentration gradient and can therefore be accomplished by simple facilitated
diffusion through GLUT2 (GLUT=glucose transporter).

key aspect of the large intestine is that it is inhabited by the so-called gut flora,
which comprises literally trillions of bacteria.5 What are these guys doing there,
other than producing foul smells, and why do we afford them such generous
shelter? There are several benefits of this:
1. Some bacteria are capable of synthesizing some vitamins, such as folic
acid, the lack of which again causes anemia, with symptoms and laboratory
findings very similar to those observed when vitamin B12 is lacking. Both folic
acid and vitamin B12 are required in the synthesis of nucleic acids. Inhibition
of DNA synthesis has the strongest impact in rapidly proliferating tissues such
as the bone marrow, which is the site of blood cell regeneration.
2. Our diet contains some substrates such as strange sugars and polysac-
charides that we cannot utilize or absorb, and which therefore travel through
the small intestine unaltered. Some bacteria can break down these substrates.6
Since the milieu in the colon is anaerobic—that is, it lacks oxygen—breakdown
will not be complete. Fairly common end products of fermentation are small
acids such as acetic or propionic acid7 Some of these products are then taken
up across the colon epithelium and utilized in our metabolism.

5 In fact, the number of bacteria in our large intestine is higher than that of our own body cells!

So, arguably, we are in fact prokaryotic ;)


6 Testing for utilization of all kind of sugars—rhamnose, raffinose, melibiose and so forth—as

well as amino acids is in fact a traditional methodology for classifying enteric bacteria.
7 Our good old friend Escherichia coli and other bacteria also produce formic acid, which they

then cleave to H2 and CO2 , which are in turn released as gases.


12 1 Introduction

villi

microvilli

blood, lymphatic vessels

Figure 1.8 Structure of the mucosa of the small intestine. Top: Schematic, show-
ing the surface structure at increasing power of magnification. The inner sur-
face of the intestine is folded, and the folds in turn have a shaggy appearance
because the mucosa is folded up again into villi. The epithelial cells that consti-
tute the mucosal surface are in turn covered by microvilli. Bottom: Villi, shown
in a stained section of the intestinal mucosa (light microscopy, left), and microvilli
on the surface of an epithelial cell (scanning electron microscopy). Below the sur-
face, you can see the folded membranes of the endoplasmic reticulum. The mi-
croscopic images have been reproduced, with permission from Roger Wagner, from
http://www.udel.edu/Biology/Wags/histopage/histopage.htm.

3. The breakdown of non-utilizable substrates in bacterial metabolism also


helps us to retrieve almost all of the water from the gut lumen. The overall
amount of fluid secreted into the digestive tract is about 7 liters per day. At
1.5 The digestive tract 13

Systemic
circulation

Liver vein

Liver

Vena portae and tributaries Liver artery

Figure 1.9 The portal circulation. The intestines receive arterial blood from the heart,
just like any other organ. The venous blood, however, is not passed back directly to
the right heart, as is the case with practically all other organs, but first reaches the liver
through the portal vein. The liver also receives a direct supply of oxygen-rich blood
through the liver artery. After passage through the liver, the blood received through
both the portal vein and the liver artery is fed into the general circulation.

the end of the small intestine, 5-5.5 liters have been reclaimed. Recovery of
this residual amount is hampered by the fact that the non-utilized solutes are
osmotically active, that is they will hold on to water and drag it down the pipe.
Utilization of these solutes in bacterial metabolism reduces the osmotic activity
of the colon content, enabling a more complete reuptake of water.8
So, you see that our intestinal bacteria are actually quite useful. On the
other hand, the bacterial fermentation also produces ammonia and other toxic
products that must be captured and disposed of by the liver. This is not a
problem in healthy individuals but can become one in persons suffering from
liver disease.

1.5.6 The portal circulation

Upon uptake, most solutes will be exported on the other side of the mucosal
cells and then find themselves in the blood stream. A peculiarity of the in-
testines consists in the fact that all blood drained from them is first passed

8 On the other hand, increasing the osmotic activity of the gut content is a straightforward

way of limiting water reuptake in order to treat constipation. A traditional drug based on this
principle is sodium sulfate (now out of fashion).
14 1 Introduction

on to the liver via the portal vein (Figure 1.9) portal circulation before being
released into the general circulation. This serves a twofold purpose:
1. It gives the liver a chance to take excess amounts of substrates—glucose,
amino acids—out of circulation and to store and process them. This serves
to maintain stable blood nutrient concentrations, which is important for the
well-being of the more sensitive and fastidious cells in the rest of the body.
2. The bacteria in the large intestine produce ammonia and other toxic
metabolites, which are cleared by the liver. In patients with liver failure, these
toxic metabolites spill over into the systemic circulation, which will among
other things lead to disturbances of cerebral function. This activity of the liver
also affects many drugs; the inactivation of drugs by the liver immediately
following intestinal uptake is known as the first pass effect.9
The liver has a particular tissue structure that enables it to exchange solutes
with the blood very efficiently. While in most organs the blood is contained in
blood vessels with clearly defined boundaries and walls, the liver has a sponge-
like structure that permits direct contact of the blood plasma with the liver
cells (Figure 1.10).

1.6 What’s next?


Digestion is only a preparatory step in metabolism. The really interesting part
starts once the substrates have been absorbed and made their way to the liver.
Their fate there will depend on the prevailing metabolic situation. The utiliza-
tion of glucose, the most important single substrate in energy metabolism, is
controlled by the hormones insulin and glucagon, both of which are secreted
in the endocrine islets of the pancreas:
1. If glucose is plentiful, typically after a meal, a significant fraction will be
retained in the liver and stored there in the form of glycogen, which is a polymer
closely similar to amylopectin. The stored glycogen is broken down again
to glucose, which stabilizes blood glucose levels during prolonged intervals
between meals. Once the glycogen stores are stocked up to the roof, glucose
and amino acids will be turned into fat, which will then be forwarded to the
peripheral fat tissue for storage.
2. If glucose is available but in high demand due to exertion, the liver will
pass on the glucose to the periphery for utilization.
3. If it is scarce, the liver will use part of the amino acids it may receive
from the intestine to turn them into glucose, which it then passes on to the
periphery.
Dietary fat is processed differently from water-soluble substrates, that is
sugars and amino acids. It is packaged into lipoproteins directly in the intestine,
9 The first pass effect, and pharmacokinetics in general, are discussed in more detail in my

Biochemical Pharmacology course notes.


1.6 What’s next? 15

Portal vein branch (from intestine) a)


Liver artery branch

To liver
vein

b)

Figure 1.10 Blood circulation and tissue perfusion in the liver. a: The liver tissue
has a honeycomb structure; each hexagon constitutes one liver lobule. The liver artery
and portal vein branches are located at the corners of each lobule. The blood seeps
out of the artery and portal vein branches, into the sinusoids of the liver lobule. It
is collected by the lobule’s central vein, which drains it toward the liver vein and the
general circulation. b: Higher power view, showing the sponge-like structure of the
liver tissue. The liver cells are stained in brown; the white space in between are the
sinusoids. While in the sinusoids, the blood gains surrounds virtually every liver cell.
The microscopic images have been reproduced, with permission from Roger Wagner,
from http://www.udel.edu/Biology/Wags/histopage/histopage.htm.

and it bypasses the liver because it is delivered into the lymphatic vessels rather
than into the blood stream.
We will consider all these processes in turn. At the end of this class, we will
not only have gained a solid grasp of these pathways, but we will also have the
tools to understand in depth what goes wrong in metabolic diseases such as
phenylketonuria, lactose intolerance, and diabetes mellitus.
Chapter 2

Refresher

This chapter attempts to summarize some essential concepts from second year
biochemistry. Feel free to skip it if you remember a thing or two from that
distant past.

2.1 How enzymes work: Active sites and catalytic mecha-


nisms
Open any textbook of biochemistry and you will be presented with an over-
whelming number of figures depicting the crystal structures of a multitude of
enzymes. Of course, the three-dimensional structures of enzymes are crucial
to their functions. Many enzymes are just regular protein molecules, composed
of nothing else than the 20 standard amino acids. If you mix of all these amino
acids in free form at, say, 1 mM each, this mixture will not have any significant
catalytic activity. This suffices to show that it is the precise arrangement of the
amino acids in the enzyme molecule that brings about its specific function.
Since the α-amino and α-carboxyl groups of the amino acids are hooked up
to each other in the polypeptide chain, they usually do not directly contribute
much to the catalytic effect of the enzyme. Instead, it is the side chains that are
directly engaged win the reaction.1 A very good example of this is chymotrypsin
(Figure 2.1). Chymotrypsin is one of the major proteases in the human digestive

16
2.1 How enzymes work: Active sites and catalytic mechanisms 17

a) O R2 O R4
H
H 2N N
N N
H H
R1 O R3 O

b) O
O

Ser 195
H 2N
H2N
O Attack on Asp 102
O
H substrate
H peptide bond

N
N His 57
His 57
N
N H H 2N
H2N
H O O Ser 195
O O

H2N
H 2N O
O
Asp 102
O
O

c) A
A−
R2 O
H
H H O
R N R2 O
R2
N C R H
H R N N
R O H R
R3 N C R
O H N C
O O H R3
O R3
O
Ser−Enzyme Ser−Enzyme Ser−Enzyme

O H
d) O OH
R2 H
H R2 O
R R2 H O
N H
R R N
N C R
H N C R N
H N C O H R
O R3 HO H
O R3
OH OH R3

Figure 2.1 Structure and mechanism of chymotrypsin. a: The peptide bond cleaved
by chymotrypsin. b: The interplay of the side chains of histidine 57, aspartate 102, and
serine 195 that results in the deprotonation of serine. All three side chains are close to
each other in the active site of the enzyme. c: The enzyme- catalized reaction. d: The
base-catalyzed reaction for comparison.

tract, where its job is to knock down large protein molecules into small peptides
that are then further processed by peptidases.
1 You will note that, nevertheless, many textbook figures give the enzyme structures as ribbon

diagrams that just show the fold of the protein backbone but omit the side chains. Therefore,
these diagrams are completely useless for understanding the catalytic mechanism of an enzyme.
18 2 Refresher

How does chymotrypsin do that? The enzyme-catalyzed reaction (Figure


2.1c) is similar to alkaline hydrolysis (Figure 2.1d). In alkaline hydrolysis, a
hydroxide ion, which is a strong nucleophile, attacks the carbon in the peptide
bond that carries a partial positive charge. In the enzyme-catalyzed reaction, a
deprotonated serine residue of the enzyme (Ser 95) plays a role similar to that
of the hydroxide ion.
Now, you know that serine normally is a neutral amino acid – its -OH group
does not spontaneously dissociate, no more than the -OH group of alcohol
does.2 The question therefore is, how does the enzyme deprotonate its own
serine side chain? This is brought about by placing the serine next to a histidine
and an aspartate inside the active center (Figure 2.1). Aspartate deprotonates
histidine, which in turn deprotonates the serine residue. This motive—asp, his,
ser—is very widespread among proteases and esterases, so much so that it is
commonly referred to as the catalytic triad. For example, the protease trypsin
and several lipases that occur in human metabolism have this motif and share
the same mechanism of catalysis.3
While with many enzymes the protein molecule and its amino acid side
chains are sufficient for catalysis, many others require coenzymes for their
catalytic activity. Very often, both active-site amino acid side chains and coen-
zymes are required. For example, amino acid transaminases (see Figure 12.4)
have a molecule of the coenzyme pyridoxalphosphate bound to the active site,
which cooperates with an active site lysine during the enzyme reaction.
Most enzymes have just one active site, or if they are multimeric one active
site per subunit. However, there are exceptions: Fatty acid synthase (see section
10.5) has as many as eight different active sites on each subunit. Multi-enzyme
complexes such as pyruvate dehydrogenase (see section 5.1) have one active
site per subunit but combine different types of subunits and enzyme activities
in one functional assembly.

2.2 Classification of Enzymes and enzyme reactions


When looking at enzyme names such as transketolase or phosphorylase, you
will note that these names don’t tell you what reactions exactly the enzymes
may catalyze, or what substrates they operate upon. A complete description
should mention the coenzymes required, the substrates and the particular
bonds in the substrates that are being severed or created. A nomenclature
that meets these criteria has been developed by the Enzyme Commission of
the IUBMB (International Union of Biochemistry and Molecular Biology). In the
IUBMB nomenclature, the enzyme transketolase bears the formidable name:
2 Otherwise,
beer would taste sour – imagine how rotten that would be.
3 Variations
of this motif, for example in the proteasome, may contain glutamic acid instead of
aspartic acid, or threonine instead of serine. These variations still contain the same functional
groups and work the same way.
2.3 Energetics of enzyme-catalyzed reactions 19

Sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetrans-


ferase (no, I can’t remember it either).
Such names, of course, are rather lengthy, and their use is not very wide-
spread. To make the tasks of tracking and bookkeeping more manageable,
these names are supplemented with numeric codes. In the IUBMB scheme,
enzymes are put into one out of six classes according to the reactions they
catalyze. These classes are:
1. Oxidoreductases. These catalyze redox reactions, frequently involving
one of the coenzymes NAD+ , NADP+ , or FAD.
2. Transferases. These bring about the transfer of functional groups, for ex-
ample a phosphate group from ATP to another metabolite, which activates
the latter and sets it up for subsequent reaction steps.
3. Hydrolases. These catalyze hydrolysis reactions, such as those involved
in digestion of foodstuffs.
4. Lyases. These enzymes effect elimination reactions that result in the
formation of double bonds.
5. Isomerases. These facilitate the interconversion of isomers. We will meet
two examples as soon as we get into glycolysis.
6. Ligases, which form new covalent bonds at the expense of ATP hydrolysis.
Of course, within each of these main classes, there are subclasses and sub-sub
classes that correspond to details of substrates and mechanisms of the enzyme
reactions. Each individual enzyme activity is assigned an individual number
within a sub-sub class, so that we wind up with a four-figure designation, which
is preceded by the letters EC (Enzyme Commission). The website http://www.
chem.qmul.ac.uk/iubmb/ gives a list of all the enzyme activities recorded by
the IUBMB classification.
One good thing about this classification is that it is rarely used. The “rec-
ommended names”, which most of the time happen to be the traditional ones,
are used instead. The other good thing is that it has a sound appreciation of
priorities. EC 1.1.1.1 is the single most important enzyme in student lifestyle –
namely, alcohol dehydrogenase, or, as IUBMB puts it, alcohol:NAD oxidoreduc-
tase. This laudable enzyme, residing in the liver, degrades ethanol, and without
it we would be drunk all the time!

2.3 Energetics of enzyme-catalyzed reactions


With each enzymatic reaction, as with any other chemical reaction, energy
comes in with two questions: (1) Will the reaction proceed at all in the desired
direction? (2) If it does, will it proceed at a sufficient rate?
The first question is decided by the free energy of the reaction, ∆G, which
when negative will make the reaction go forward. The second question depends
on the activation energy, ∆G∗ , which forms a barrier between the two states of
20 2 Refresher

Barrage
Turbine

Figure 2.2 The Kochelsee-Walchensee hydroelectric power system. The Walchensee


is situated above the Kochelsee. Artificial conduits connect the two lakes and also drain
other lakes into the Walchensee.

the reactants. The very short-lived, energy-rich state at the top of this barrier is
called the transition state. Enzymes can make a big difference to the activation
energy ∆G∗ and thus accelerate reactions, but they cannot change the free
energy ∆G—and therefore, the direction—of the reaction.

2.3.1 A simile

The different roles of ∆G and ∆G∗ in biochemical reactions can be illustrated


with a simile. Figure 2.2 below shows two lakes in the German Alps. The
Walchensee4 is situated 200 m above the Kochelsee. A conduit was dug across
the barrier between these two lakes to make the water flow downhill and drive
a hydroelectric turbine. Additional tunnels drain other lakes and rivers to
enhance the supply of water to the Walchensee.
Driven by the considerable hydrostatic pressure accruing during the flow
downhill, this system generates quite a bit of electrical energy. It can be likened
to a metabolic pathway:
• The difference in altitude between the reservoirs is similar to the free
energy difference, ∆G, between two metabolites.
• The height of the barrier between the two lakes is similar to the activation
energy (∆G∗ ) of the transition between two metabolites. Enzymes facili-
tate the interconversion of metabolites by creating “tunnels” that bypass
the activation energy barrier between them.
• In the hydro-electric system, tunnels can be opened or shut by barrages to
accommodate different amounts of rainfall or of electrical energy required.
Likewise, enzymes have switches to regulate their activity to allow for
adjustments in the flow along metabolic pathways.

4 Der See=German for the lake, die See=German for the sea
2.4 The role of ATP in enzyme-catalyzed reactions 21

• Like the conduits, enzymes can facilitate the flow and (typically) control
its rate but not its direction. The direction of the flow will depend solely
on the difference in altitude (∆G).
It is usually best not to take analogies too far, because otherwise they be-
come obfuscating rather than enlightening. Here are some dissimilarities:
• All tunnels are basically alike. In contrast, each enzyme needs a specific
“trick” or catalytic mechanism suited to the specific metabolites it is deal-
ing with. Investigating the catalytic mechanisms of individual enzymes is
an important part of biochemistry.
• The energy level of a pool in the hydro-electric system is completely
described by its altitude. However, the free energy of a metabolite is
dependent on its concentration; the lower the concentration, the lower
the free energy.
• Although enzymes cannot revert an endergonic reaction (that is, a reaction
with ∆G > 0) in isolation, they can make it go uphill by coupling it to
another one that is exergonic, so that the overall ∆G becomes negative.
• Finally, water will never spontaneously flow uphill – molecules, however,
do occasionally move spontaneously to somewhat higher energy levels.
If molecules are offered two states with different energy levels, such as two
different conformations or two different tautomeric states, they will sponta-
neously distribute between them and establish equilibrium. In equilibrium,
the relative occupancy of these two states will solely depend on the energy
difference between them:
n1
= e− RT
∆G
(2.1)
n2
where n1 and n2 represent the numbers of molecules in the high and low
energy states, respectively. (R is the gas constant, whereas T is the absolute
temperature.)
The above equation applies to the distribution of the initial and the final
states of a reaction. It also applies to the distribution between molecules be-
tween the initial state and the transition state of a reaction. The tendency of
molecules to spontaneously assume states of higher energy explains that chem-
ical reactions can occur at all, even though the energy level of the transition
state is always higher than those of the initial and final states. However, the
higher the activation energy, the more rarified the transition state will become.
The number of molecules that can make it across the barrier thus becomes
smaller, and the reaction slower with increasing activation energy.

2.4 The role of ATP in enzyme-catalyzed reactions


The most common exergonic reaction that is utilized by enzymes to drive en-
dergonic ones is the hydrolysis of the phosphodiester bonds in ATP. While this
22 2 Refresher

O O

H2N CH
CH2 + O O O

CH2 O P O P O P O Adenosine
O O O
O O

HO O

H2N CH O O
CH2 + O P O P O Adenosine
CH2 O O
O
O O P O
O

NH3

HO O O O
+ O P O P O Adenosine
H2N CH
O O
CH2
CH2 O
+ O P O
O NH2
O

Figure 2.3 The catalytic mechanism of glutamine synthetase. The terminal phos-
phate is first transferred to glutamate to form a carboxyphosphate. In this energy-rich
intermediate, the phosphate is a good leaving group and is easily substituted by am-
monia.

is a general principle in enzymology, it is important to understand that there is


no equally general chemical mechanism of ATP utilization: Each enzyme needs
to find its own way of actually linking it to the reaction it needs to drive. As an
example, here is how glutamine synthetase does it. This enzyme uses ATP to
produce glutamine from glutamate and ammonia (Figure 2.3).
While the net turnover of ATP is hydrolysis, the phosphate group is actually
first transferred to the substrate to create an intermediate product, glutamate-
5-phosphate. The phosphate group in this compound—a mixed anhydride—is
a very good leaving group, which facilitates the subsequent substitution by
2.5 Regulation of enzyme activity 23

ATP (cosubstrate)
Fructose-6-P

ADP (regulator)

Figure 2.4 Structure of the enzyme phosphofructokinase (shown in wire-frame) with


bound substrate (fructose-6-phosphate), cosubstrate (ATP) and allosteric regulator
(ADP), all shown in space-filling mode. Substrate and cosubstrate reside in the active
site, while ADP binds to a separate regulatory site.

ammonia. Therefore, the utilization of ATP is an integral part of the reaction


mechanism of this enzyme. We will see additional examples of ATP usage in
enzyme catalysis in the remainder of this course.

2.5 Regulation of enzyme activity


The enzyme phosphofructokinase catalyses the following reaction:

Fructose-6-phosphate + ATP → Fructose-1,6-bisphosphate + ADP (2.2)

This reaction occurs as an early step in the degradation of glucose, which


ultimately serves to replenish ATP from ADP and phosphate. It therefore makes
sense that phosphofructokinase should be stimulated by ADP.
To accomplish this stimulation, ADP binds to a distinct site on the enzyme
that is far away from the active site; it therefore clearly does not directly
participate in the reaction (Figure 2.4). Instead, the binding of ADP changes
the conformation of the entire enzyme molecule. The change will also affect
the active site and enhance the efficiency of catalysis there. This mode of
action is known as allosteric regulation and is exceedingly common. Allosteric
24 2 Refresher

a) c)

R A R A

Active Inactive
conformation conformation

Allosteric regulation and cooperativity


b)

d)
P
Allosteric Allosteric
activation inhibition
Inhibition by phosphorylation

Figure 2.5 Enzyme regulation by allosteric effectors and by phosphorylation. a: The


regulatory binding site for the allosteric effector (R) is distinct from the catalytic active
site (A) of the enzyme. The enzyme can assume two distinct conformations, and both
the regulatory site and the active site are affected by the transition between them. The
enzyme is active only in one of these conformations. b: An allosteric inhibitor binds
to a regulatory site in the inactive conformation; the energy of binding favours this
conformation. An allosteric activator binds and stabilizes the active conformation.
c: Most allosteric enzymes are multimeric. The conformational transition affects the
interfaces between the subunits, thereby enforcing the transitions of all subunits to
occur synchronously or cooperatively. d: Regulation by phosphorylation also works
by selective stabilization of one conformation. Like allosteric regulation, it can be
inhibitory (as shown here) or stimulatory.

effectors can be either stimulatory, as ADP is in this example, or inhibitory. As


an example of the latter, ATP not only binds to the active site but also acts as
an allosteric inhibitor of phosphofructokinase – which again makes sense in
the context of overall physiological regulation.
The workings of allosteric regulation are schematically depicted Figure 2.5.
The enzyme has two possible conformations that are in equilibrium with each
other. An allosteric activator will bind selectively to the regulatory site as it oc-
curs in the active conformation and thereby shift the equilibrium towards this
conformation. Conversely, an inhibitor would bind selectively to the inactive
conformation and thereby stabilize it. As you can see, activators and inhibitors
may share the same regulatory site; this is the case in the above example of
phosphofructokinase with ATP and ADP. Note, however, that phosphofructoki-
nase has additional allosteric sites that permit regulation by other effectors (see
Figure 7.4). Although it is not theoretically necessary, it seems that all allosteric
enzymes occur as oligomers. In Figure 2.5c, a dimeric enzyme is shown, which
is not uncommon, but often the number of subunits is considerably higher.
Their oligomeric nature enables enzymes to behave cooperatively, that is to
react more sensitively to changes in effector concentration.
2.5 Regulation of enzyme activity 25

Another important means of enzyme regulation is through protein phos-


phorylation. This occurs by protein kinases, which transfer a phosphate group
from ATP to a specific protein side chain on the regulated enzyme. The mecha-
nism of regulation by phosphorylation is not really that different from allosteric
regulation. The only difference is that in this case the regulator is more stably
attached, so that the regulatory effect will last longer. Like allosteric regulation,
it can be inhibitory (as shown in Figure 2.5d) or stimulatory. Many enzymes are
subject to regulation both by allosteric effectors and by phosporylation.
Note that the functional effect to any specific allosteric regulator, and of
phosphorylation as well, depends entirely on the enzyme in question. For
example, while ATP inhibits phosphofructokinase, it activates the function-
ally opposite enzyme fructose-1,6-bisphosphatase (section 7.3.2). Likewise,
phosphorylation by protein kinase A inhibits glycogen synthase but activates
glycogen phosphorylase (section 8.4).
While all mechanisms discussed so far modulate the activity of existing
enzyme molecules, the overall of an enzyme may also be varied by changing
the abundance of enzyme molecules. Firstly, the transcription of the gene
encoding the enzyme in question can be turned on or off. This mechanism is
employed by many hormones, in particular steroid hormones such as cortisone
and by thyroid hormones. Enzyme molecules can also be tagged for accelerated
proteolytic degradation. Similarly, the stability of the enzyme messenger RNAs
encoding specific enzymes can be regulated up or down, with corresponding
effects on the abundance of the enzyme molecules. Hormones may affect the
activity of an enzyme at more than one level. For example, insulin increases
the activity of glycogen synthase by way of transcriptional induction, increased
mRNA stability, and protein phosphorylation.
Chapter 3

Glycolysis

Glucose is a key metabolite in human metabolism, and we will spend a good bit
of time on the several pathways that are concerned with the utilization, storage,
and regeneration of glucose.

3.1 Overview of glucose metabolism


There are five major pathways of glucose metabolism:
1. Glycolysis, which accomplishes the degradation of glucose to pyruvate.
Its main purpose is the generation of energy (ATP). Glycolysis generates some
ATP directly, and a lot more indirectly through the subsequent oxidation of
pyruvate. The need for ATP is universal, so that the glycolytic pathway is found
in every cell of our body.
2. The hexose monophosphate shunt. This pathway also breaks down glu-
cose, but the main product is not ATP but NADPH. NADPH is universally needed
as a reducing agent, so that this pathway is ubiquitous, too.
3. Glycogen synthesis. Glycogen is a polymeric storage form of glucose, not
unlike starch, which is found in plants. This pathway is quantitatively most
important in the liver and striated muscle,1 although some is found in in other
tissues also. The glycogen synthesized is retained within the same cell.
1 Striated muscle comprises skeletal muscle and heart muscle. The second major type of

muscle tissue is smooth muscle, which occurs in blood vessels and internal organs and is under
autonomous control.

26
3.2 The place of glycolysis in glucose degradation 27

1 2 3 Fatty acids,
Glucose Pyruvate Acetyl-CoA
triacylglycerol
H2O
4
CO2
“H2“
O2 ADP + Pi
5
ATP
H2O

Figure 3.1 Overview of glucose degradation. 1: Glycolysis; 2: Pyruvate dehydroge-


nase; 3: Fatty acid synthesis; 4: Citric acid cycle; 5: Respiratory chain. The "H2" that is
produced in the citric acid cycle and oxidized in the respiratory chain is not gaseous
but bound to co-substrates.

4. Breakdown of glycogen to glucose, like glycogen synthesis most impor-


tant in liver and muscle. This pathway is activated if the current external supply
of glucose is low, as it is between meals. In the liver, the glucose generated
from glycogen is released into the general circulation. Sceletal muscle cells
may utilize the glucose themselves, for the purpose of ATP synthesis and then
muscle activity (contraction).
5. Gluconeogenesis. This pathway turns pyruvate derived from amino acids
into glucose; it thus is essentially the reversal of glycolysis. It, too, is activated
in times of low external glucose supply. The amino acid substrates may be ob-
tained from a protein-rich diet—for example if we feast on meat exclusively—or
by breaking down internal protein, mainly in skeletal muscle. Gluconeogenesis
occurs in the liver and in the kidneys.
We will consider all these pathways in their turn, starting with glycolysis.

3.2 The place of glycolysis in glucose degradation

Figure 3.1 gives an overview of the steps involved in the complete degradation
of glucose. It is evident that, in the overall process, glycolysis is only the
first step. The pyruvate it generates is turned into acetyl-CoA by pyruvate
dehydrogenase. Acetyl-CoA is completely degraded in the citric acid cycle and
the respiratory chain. While some ATP is generated at each of these stages,
most of it is produced in the respiratory chain.
If glucose is available in excess of immediate needs and of glycogen storage
capacity, it will still be broken down by glycolysis and pyruvate dehydrogenase
to acetyl-CoA. However, acetyl-CoA will then be fed into fatty acid synthesis,
which occurs in the liver and the fat tissue.
28 3 Glycolysis

3.3 Reactions in glycolysis


Glycolysis involves 10 enzymatic reactions, summarized in Figure 3.2:
1. The phosphorylation of glucose at position 6 by hexokinase,
2. the isomerization of glucose-6-phosphate to fructose-6-phosphate by
phosphohexose isomerase,
3. the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate
by phosphofructokinase,
4. the cleavage of fructose-1,6-bisphosphate by aldolase. This yields two
different products, dihydroxyacetone phosphate and glyceraldehyde-3-phos-
phate,
5. the isomerization of dihydroxyacetone phosphate to a second molecule
of glyceraldehyde phosphate by triose phosphate isomerase,
6. the dehydrogenation and concomitant phosphorylation of glyceralde-
hyde-3-phosphate to 1,3-bis-phosphoglycerate by glyceraldehyde-3-phosphate
dehydrogenase,
7. the transfer of the 1-phosphate group from 1,3-bis-phosphoglycerate to
ADP by phosphoglycerate kinase, which yields ATP and 3-phosphoglycerate,
8. the isomerization of 3-phosphoglycerate to 2-phosphoglycerate by phos-
phoglycerate mutase,
9. the dehydration of 2-phosphoglycerate to phosphoenolpyruvate by eno-
lase, and finally
10. the transfer of the phosphate group from phosphoenolpyruvate to ADP
by pyruvate kinase, to yield a second molecule of ATP.

3.4 Mechanisms of enzyme catalysis in glycolysis


Metabolic reactions are catalyzed by enzymes. Enzymes are not magicians but
sophisticated catalysts, and their chemical mechanisms are often understood
quite well, at least in principle. We will look at a few selected examples that il-
lustrate various mechanisms of catalysis that occur similarly in other metabolic
pathways, too.

3.4.1 Hexokinase
Our first example is hexokinase, which carries out the first reaction in the gly-
colytic pathway. This type of reaction—the transfer of the terminal phosphate
group from ATP onto a hydroxyl group on the substrate—is a very common
reaction in biochemistry, and we will see many more examples in this class.
The mechanism of phosphorylation is always the same, so it suffices to discuss
it once.
Most reactions that involve the transfer of a phosphate group from ATP
to something else are exergonic, that is they are energetically favourable. For
example, the hydrolysis of ATP—which is the transfer of the phosphate group
3.4 Mechanisms of enzyme catalysis in glycolysis 29

O
Glucose Glucose-6-P Fructose-6-P
HO P O-
O
HO O HO P O-
O OH
O ATP ADP O O
OH OH
OH
OH OH OH OH OH
OH 1 OH 2 OH

ATP
1,3-Bisphosphoglycerate
O
3
ADP
HO P O- GAD-3-P DHAP
O O OH
O O O
+ +
OH NADH+H NAD OH O HO P O- HO P O-
+ O O
O
O 6 O O 4
Pi OH
HO P O- HO P O- HO P O- OH
O O O OH
5
Fructose-1,6-bis-P
ADP
7
ATP
2-Phosphoglycerate Phosphoenolpyruvate Pyruvate
O -
O -
O - O-
O O O -
O O -
O
10
OH O P O O P O O
8 OH 9 OH
ADP ATP
O OH
HO P O-
O 3-Phosphoglycerate

Figure 3.2 The glycolytic pathway. Enzymes: 1, Hexokinase; 2: Phosphohexose


isomerase; 3: Phosphofructokinase; 4: Aldolase; 5: Triose phosphate isomerase; 6:
Glyceraldehyde-3-phosphate dehydrogenase; 7: Phosphoglycerate kinase; 8: Phospho-
glycerate mutase; 9: Enolase; 10: Pyruvate kinase. Reversible reactions are indicated
by double arrows, irreversible ones by single arrows. The reversible reactions are part
of both glycolysis and gluconeogenesis; irreversible ones require workarounds in glu-
coneogenesis. (Abbreviations: -P, phosphate; GAD-3-P, glyceraldehyde-3-phosphate;
DHAP, dihydroxyacetonephosphate)
30 3 Glycolysis

a) O O O
b)
O P O P O P O Adenosine
Arg
R X O O O Lys HN NH

Mg++ +
NH3
+
O O O NH3 O O O
R X P O O P O P O Adenosine O P O P O P O Adenosine

O O O R X O O O

Figure 3.3 Mechanism of phosphate group transfer from ATP. a: The phosphorus in
the center of the terminal phosphate group is attacked by a nucleophile, which often is
an anion (R–X– ) but can also be a free electron pair. Attack is inhibited by the negative
charges on the oxygen atoms around the phosphorus. This electrostatic repulsion
is responsible for the high activation energy barrier of the uncatalyzed reaction. b:
ATP is activated towards nucleophilic attack through electrostatic shielding both by
magnesium and by positively charged amino acid side chains in the active site of the
enzyme.

to water—has a free energy of −35 J/K mol. It is interesting to note then that,
despite the abundance of water, ATP is quite stable in solution. This indicates
that there must be a high activation energy barrier that resists cleavage of the
phosphodiester bond.2
This energy barrier is due to the negative charges within the phosphate
group that shield the central phosphorus from nucleophiles that likewise are
negatively charged. Accordingly, to lower this barrier, kinases provide com-
pensating positive charges within the active sites, which engage the negative
charges on the ATP molecule and thereby facilitate nucleophilic attack (Figure
3.3).
The intracellular concentration of glucose will be in the low millimolar range,
whereas water is present at a concentration of more then 30 mol/l. Apart from
the task of activating ATP, then, hexokinase also must ensure that the activated
ATP does indeed react with the hydroxyl group on the glucose molecule but
not of water. How is this specificity accomplished?
Like many other enzymes, hexokinase exists in an open and a closed confor-
mation. It binds its substrate and cosubstrate, glucose and ATP, in the open
state, whereupon it changes its conformation to the closed state. This change
is necessary for the enzyme’s catalytic activity, and it also is accompanied by
the expulsion of water from the active site (Figure 3.4). In this way, the only
hydroxyl group that remains in the vicinity of the activated ATP will be that
on the C6 of glucose. However, if we offer the enzyme xylose as a substrate
2 If you are unclear about the difference between free energy and activation energy of a chemical

reaction, have a look at section 2.3.


3.4 Mechanisms of enzyme catalysis in glycolysis 31

a) b) H2C OH
O
OH
OH OH
Glucose OH

H
OH
O
OH
OH OH
OH
Xylose + water

Figure 3.4 How hexokinase circumvents ATP hydrolysis. a: After binding of its
substrate glucose (red/spacefill) and its cosubstrate ATP (not shown), hexokinase
(blue/wire- frame) adopts a closed conformation, in which substrate and cosubstrate
are buried within the enzyme. In this way, water is excluded from the active site and,
hence, from the reaction. b: If xylose is used instead of glucose, one water molecule can
squeeze along with it into the active site and then react with ATP, leading to hydrolysis.

instead of glucose, we can fool it into hydrolyzing ATP rather than phospho-
rylating the sugar. Xylose looks exactly like glucose with respect to the ring
(C1 – C5), so it is able to bind to the active site of hexokinase and induce the
activating conformational change. However, because it lacks the C6 atom with
its attached hydroxyl group, it leaves space within the active site for one water
molecule (Figure 3.4b), and it is this water molecule that will react with the
activated ATP in this case.

3.4.2 Phosphohexose isomerase

The mechanism of phosphohexose isomerase is a good example of acid-base


catalysis. The active site contains two basic groups, one of which is protonated
at the start of the reaction (Figure 3.5). Several successive protonations and
deprotonations lead first to ring opening, then to shifting of the double bond
from the aldo- to the keto-form, and finally to ring closure within the fructose-
6-phosphate.

3.4.3 Glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase provides a straightforward exam-


ple of another type of enzyme catalysis, known as covalent catalysis. This
reaction mechanism is very common in dehydrogenation reactions, for exam-
32 3 Glycolysis

CH2O-Pho. Glucose-6-P
HC O
2
HC OH CH B+
H CH2O-Pho.
OH C C OH H
H HC O
OH
HC OH CH
1 H 3
B+ OH C
H
C O H B:
CH2O-Pho
OH
H - HC OH
B H H
HC OH C
OH C O
H
OH H+
- -
B B

5 4
:B CH2O-Pho
B:
CH2O-Pho
HC O H HC OH
H2 H
HC OH HC OH C
C
CH2O-Pho O
OH C OH OH C
H H+
H
HC O
H2 O O H
H
HC OH C H B B
OH C OH
H
OH Fructose-6-P

Figure 3.5 The catalytic mechanism of phosphohexose isomerase. 1: The active site
contains 2 bases in strategic positions. 2: Glucose-6-phosphate bound the active site,
and the ‘electron-hopping’ steps that lead to ring opening. 3-5: Subsequent reaction
steps that lead to migration of the keto group and ring closure of fructose-6-phosphate.

ple in the citric acid cycle and in the β-oxidation of fatty acids. The reaction
goes through the following steps (Figure 3.6):
1. The substrate binds to the active site, where NAD+ is already bound.
2. A deprotonated cysteine thiol group (–S– ) in the active site performs a
nucleophilic attack on the aldehyde carbon, which yields a tetrahedral
intermediate state in which the enzyme and the substrate are covalently
bound to each other – hence the term “covalent catalysis”.
3. The intermediate transfers 2 electrons and a proton to NAD+ , yielding
NADH and a thioester.
4. NADH leaves and is replaced by NAD+ .
5. The thioester is cleaved by a phosphate ion, again by nucleophilic attack.
6. The product (1,3-bisphosphoglycerate) leaves, and the enzyme is restored
to its original state.
The redox cosubstrate used by glyceraldehyde-3-phosphate dehydrogenase,
nicotinamide adenine dinucleotide (NAD+ ), is the major acceptor of hydrogen
3.4 Mechanisms of enzyme catalysis in glycolysis 33

a) O O O O
P P
H O O O
H O O O O O

OH R OH R
O O

O P O O P O

O O

b)
1 2 3
NAD+ NAD+ H NAD
+
H O
H O
- - C O
S S S
R R
R
+ + +
HB HB HB

6 5 4
+ + NADH
NAD
NAD
O O O
C C +
S—H S O P OH S NAD
R R
O
:B + +
HB HB

O
O
O P OH

R O

Figure 3.6 The catalytic mechanism of glyceraldehyde-3-phosphate dehydrogenase.


a: All the action occurs at the substrate’s aldehyde group. For simplicity, the remain-
der is therefore abbreviated as R in b. The substrate glyceraldehyde-3-phosphate is
shown on the left, the product 1,3-bisphosphoglycerate on the right. b: The reaction
mechanism. B represents a basic residue in the active site. See text for details.

abstracted from substrates throughout glycolysis and the citric acid cycle. Its
structure and its reduction by hydrogen are shown in Figure 3.7. As you can
see, all the action occurs at the nicotinamide moiety – the adenosine part is
completely out of the picture, as far as the redox chemistry is concerned. Why,
then, is it there at all? One answer is that it serves as a “tag”, an identifier
that enables the coenzyme to interact with a defined set of enzymes. There
34 3 Glycolysis

a) O b)

NH2 O

+ E S C H
N O
R H
C O C
HC NH2
O
HC +
CH
O P O N
OH OH
O R
NH2
O P O
O
O N
N E S C
H H O
C N R
O N
HC NH2

HC CH
N
OH OR R=H: NAD+
R=phosphate: NADP+ R

Figure 3.7 Reduction of nicotinamide adenine dinucleotide (NAD+) by


glyceraldehyde-3- phosphate dehydrogenase. a: Structure of NAD+ and NADP+ .
The redox-active nicotinamide moiety is at the top. The two molecules differ solely by
the lack or presence of a phosphate group at the lower ribose ring. b: The electrons
and the hydrogen are transferred to the C4 of the nicotinamide; the electrons then
redistribute within the ring.

is a second, very similar coenzyme, NADP+ , which performs the same redox
chemistry but has a different tag with an extra phosphate group and is used by
a different set of enzymes. The reasons for this are discussed in a later chapter
(see section 9.3).
It is also interesting to note that, as in many other cosubstrates, the tag
consists of a nucleotide instead of a peptide. This likely hearkens back to
the ancient RNA world, in which all catalytic functions are supposed to have
been performed by RNA enzymes instead of proteins. These RNA enzymes are,
for the most part, now extinct, having been replaced by presumably superior
protein enzymes. However, cosubstrates can’t evolve as easily as enzymes,
since they interact with many different enzymes and are therefore are subject
to many more evolutionary constraints; they thus remain frozen in time, like
molecular fossils.

3.4.4 Pyruvate Kinase


The mechanism of pyruvate kinase is similar to that of hexokinase – except that
the reaction proceeds the other way, producing ATP rather than consuming it.
Yet, both reactions are irreversible. How come?
3.5 Energy-rich functional groups in substrates of glycolysis 35

NH2

N
N
O O O

O P O O P O P O C N
O N
HO O + O O
H
CH2
O
OH OH

HO O H HO O
CH2 CH3
O O

Figure 3.8 Mechanism of the pyruvate kinase reaction. The enolpyruvate formed
intermittently tautomerizes to pyruvate; it is this step that drives the reaction in the
indicated direction.

The intermediate product of the pyruvate kinase reaction, after transfer of


the phosphate group from position 2 to ADP, is enolpyruvate. Getting rid of the
phosphate group enables the enol group to rearrange itself into a keto group
(Figure 3.8). This second step of the reaction is strongly exergonic, and it thus
pulls the overall equilibrium of the reaction over to its side. No such exergonic
step occurs with hexokinase, which explains the difference in ∆G.

3.5 Energy-rich functional groups in substrates of glycol-


ysis
You will have heard that the most abundant and important energy-rich metabo-
lite in the cell is ATP. Within this molecule, the energy is stored in the energy-
rich phosphodiester bonds. Cleavage of these bonds is exergonic, and the
energy released in the cleavage drives the various reactions and processes pow-
ered by ATP. Conversely, in creating ATP from ADP, we require energy to form
a new phosphodiester bond. We just saw where this energy is derived from in
phosphoenolpyruvate, and we thus may say that the enolphosphate group is
another energy-rich group.
The first ATP in glycolysis is formed by cleavage of the carboxyphosphate
mixed anhydride in 1,3-bisphosphoglycerate, and so we may infer that such
mixed anhydrides are energy-rich groups as well. Indeed, the same group also
occurs in acetylphosphate and in succinylphosphate, and both of these are
capable as well to drive the formation of a phosphodiester bond in ATP or GTP.
If we look again at Figure 3.6, we see that the mixed anhydride formed from
ionic phosphate and a thioester bond. Since ionic phosphate represents a low-
energy form of phosphate, it follows that the energy required for the formation
of the mixed anhydride must be derived from the thioester, whose formation
36 3 Glycolysis

in turn is driven by the partial oxidation of carbon 1, which changes from the
aldehyde form to the more highly oxidized carboxylate form.
In summary, the following functional groups can provide sufficient energy
to drive the synthesis of ATP:
1. thioesters – when cleaved,
2. aldehydes – when oxidized to carboxylic acids,
3. carboxyphosphates – when cleaved, and
4. enolphosphates – when cleaved.

3.6 Function of glycolysis under anaerobic conditions


Two molecules of ATP are expended in the initial phosphorylation steps (1 and
3 in Figure 3.2). ATP is gained in steps 7 and 10. Since all of the steps from 6 to
10 occur twice per molecule of glucose, the net balance is a gain of two moles
of ATP per mole of glucose – a very modest number indeed, considering that
the overall yield in complete oxidative degradation is around 30 moles of ATP.
Still, degradation of glucose to pyruvate is a viable source of energy, and it is
the major one that operates in our tissues while oxygen is in short supply, as is
the case in skeletal muscle during maximal exercise such as a 100 meter dash.
To make anaerobic, that is oxygen-free glycolysis feasible, we have to solve
one problem. In the glyceraldehyde-3-phosphate dehydrogenase reaction, a mo-
lecule of NAD+ is consumed and converted to NADH. Under aerobic conditions,
that is when oxygen is available, NADH is reverted to NAD+ in the respira-
tory chain. However, under anaerobic conditions, we need another means to
regenerate NAD+ .
This problem is overcome by the hydrogenation of pyruvate to lactate by
lactate dehydrogenase (Figure 3.9a). Even then, as you know from experience,
this maximal level of exertion cannot be kept up for long. We will soon have
to slow down as exhaustion and then pain set in. Exhaustion is due to the
depletion of ATP and of glucose, while pain is due to the accumulation of
lactate in the tissues and the blood.3
Some cells in the human body, most notably the erythrocytes (red blood
cells), rely entirely on anaerobic glycolysis for ATP production, since they lack
the ability to oxidize pyruvate. Anaerobic glycolysis is a common pathway of
energy production not only in animals but also in microbes, for example in
baker’s yeast. These face the same problems as human cells do, that is they
need to regenerate NAD+ and dispose of the acid. The latter task is even more
pressing for them than for our us, since they don’t have a blood circulation to
3 Measurement of the blood lactate concentration is performed in sports medicine to gauge

the capacity of a trained athlete to sustain aerobic rather than anaerobic metabolism during
prolonged exertion. The anatomical correlate of endurance is not so much the build-up of muscle
tissue but the extent of its vascularization, that is the abundance of capillaries in the tissue. A
high density of capillaries ensures good oxygen supply.
3.7 Transport and utilization of glucose 37

a) Pyruvate Lactate
O OH O OH
NAD+
NADH+H+
O HC OH

CH3 CH3

b) Pyruvate
O OH +
CO2 NADH+H+ NAD

O HC O H2C OH

CH3 CH3 CH3

Figure 3.9 Regeneration of NAD+ for glycolysis under anaerobic conditions. a: The
lactate dehydrogenase reaction is utilized by mammalians. b: Ethanolic fermentation
occurs in yeast. Pyruvate generated by glycolysis is decarboxylated to acetaldehyde.
Reduction of the latter regenerates NAD+ and yields ethanol, which is less toxic than
both the aldehyde and the lactic acid.

carry away, dilute and buffer the excess acid. One effective strategy, then, is to
chemically degrade it. This is the biological purpose of ethanolic fermentation
(Figure 3.9b).

3.7 Transport and utilization of glucose in the liver and


in peripheral cells
The liver has a special role in many metabolic processes, and prominently so
in glucose metabolism. Recall that all the glucose that is taken up from the
small intestine must pass through the liver first before it can reach any other
tissue. The fraction of glucose retained by the liver is regulated depending on
the metabolic situation:
• If blood glucose is high, the liver extracts a relatively large fraction and
uses it for conversion to glycogen or for conversion to triacylglycerol (fat).
• If blood glucose is low, the liver will only extract small amounts of glucose;
in fact, release of glucose by the liver will exceed its uptake under these
circumstances.
This latter behaviour is different for example from the brain, which un-
abashedly extracts glucose at both high and low glucose levels. This difference
between the liver and other tissues is implemented at two stages: (1) The uptake
of glucose into the cells, and (2) the phophorylation of glucose to glucose-6-
phosphate.
We have discussed before that glucose passes membranes by way of special-
ized transporter proteins. Except in the luminal membranes of the gut and of
38 3 Glycolysis

+
Figure 3.10 Facilitated diffusion, a form of protein-mediated membrane transport.
Typically, substrate binding and dissociation are fast as compared to the conforma-
tional change in the transporter protein that leads to release of the substrate on the
far side of the membrane.

the kidney tubules, these transporter proteins operate not by active transport
but by facilitated diffusion, that is they simply speed up transport of glucose
along its concentration gradient (Figure 3.10). Although such transporters are
not enzymes, there is one similarity: With both enzyme reaction and with fa-
cilitated diffusion, substrate binding and dissociation are much faster than the
action of the protein – the enzyme reaction, or in this case the conformational
change required for translocation. Therefore, like simple enzymes, facilitated
transport obeys Michaelis-Menten kinetics:

[S]
V = Vmax (3.1)
KM + [S]

You may recall (or else infer from the above equation) that the affinity of the
protein for the substrate, which is represented by KM , controls the dependency
of the activity of on the substrate concentration. In most tissues, the glucose
transporters have a low KM , which means that each transporter molecule is
always operating at full speed,4 irrespective of the glucose concentration. In
contrast, the transporter subtype found in the liver (GLUT2) has a higher KM ,
so that the rate of transport is reduced at low glucose concentration.
A similar difference is observed at the stage of glucose phosphorylation.
The liver has a special enzyme called glucokinase, which performs the same
reaction as does hexokinase but differs from the latter by a higher KM value.
Accordingly, phosphorylation will proceed at reduced rate at a low level of
blood glucose (Figure 3.11), and most of the glucose will be allowed to pass
the liver and make its way into the general circulation. Conversely, at high

4 This does not mean that the overall extraction of glucose always goes at full speed in all

tissues. Regulation of glucose uptake in many tissues is mediated by insulin, which changes
the overall number of available transporters but not the activity of the individual transporter
molecule (See section 13.1.2).
3.7 Transport and utilization of glucose 39

Hexokinase (most tissues)

Glucokinase
(liver)
V/Vmax

5 10 15 20 25
Glucose (mM)

Figure 3.11 Activities of Hexokinase and of glucokinase as a function of glucose con-


centration. The physiological range of the glucose concentration is 5-9 mM. Hexokinase
will always be fully active, whereas the activity of glucokinase varies with the substrate
concentration.

concentration, a higher amount of glucose will be extracted by the liver and


fed into synthesis of glycogen or fatty acids. The kinetic properties of both
transport and phosphorylation therefore contribute to the regulatory function
of the liver in glucose metabolism.
Chapter 4

Catabolism of sugars other than glucose

Starch is the most important carbohydrate in our diet. As we have seen, starch
consists of glucose, which is therefore the most important dietary monosaccha-
ride. Other quantitatively important sugars are:
1. Lactose (milk sugar) is a disaccharide of glucose and galactose and is
found in milk (surprise!).
2. Sucrose is a disaccharide of glucose and fructose and is found in many
plants and fruit, most prominently in sugar cane and sugar beet.
3. Fructose, as the free monosaccharide, is also found in significant amounts
in the diet, both in fruit and as a sweetener.
4. Sorbitol is a sugar alcohol, that is the carbonyl group found in fructose or
glucose is reduced to a –CHOH group. It is used as a sweetener but also
occurs naturally in the diet.
5. Nucleic acids contain ribose and deoxyribose.
Ribose is part of the hexose monophosphate shunt and will be covered in the
corresponding chapter. Here, we will focus on the first three sugars.
The main motif in the metabolism of these sugars is economy: Instead of
completely separate degradative pathways, there are short adapter pathways
that funnel them into the main pathway of carbohydrate degradation, that is
glycolysis. An overview of these pathways is given in Figure 4.1.

40
4.1 Metabolism of sucrose and fructose 41

UDP-Galactose UDP-Glucose

Galactose Galactose 1-P Glucose 1-P

Lactose Glucose Glucose 6-P


Sucrose
Fructose Sorbitol

Fructose 1-P Glyceraldehyde


Glyceraldehyde 3-P/
Dihydroxyacetone-P

Pyruvate

Figure 4.1 Metabolism of lactose / galactose, sucrose / fructose, and of sorbitol


(overview). Short adapter pathways funnel these substrates into glycolysis.

4.1 Metabolism of sucrose and fructose

Sucrose is produced from sugar cane and sugar beet, where it is found in very
high concentrations (15-18%). In our “healthy” western diet, it may amount to
as much as 20% of dietary carbohydrate. Sucrose contains glucose and fructose
joined in a β-glycosidic bond (Figure 4.2).
Hydrolytic cleavage of sucrose, like that of of maltose, occurs at the surface
of the intestinal epithelial cells. The enzyme responsible is β-fructosidase, also
named sucrase. Both sugars are then taken up by specific transport: Glucose by
the SGLT1 transporter, and fructose by the GLUT5 transporter, which is named
after glucose but in fact is more active on fructose than on glucose.
Fructose degradation, sometimes called fructolysis, is carried out in the
liver. In the first step, fructose is phosphorylated by fructokinase, which uses
ATP as a cosubstrate. This yields fructose-1-phosphate. The latter is then
cleaved by aldolase B, which is found mainly in the liver, in keeping with the
liver’s prominent role in fructose degradation. The products of this reaction
are dihydroxyacetone phosphate, which is a metabolite in glycolysis, and glycer-
aldehyde. Finally, glyceraldehyde is phosphorylated by glyceraldehyde kinase.
This yields glyceraldehyde-3-phosphate, which again is an intermediate of gly-
colysis (Figure 4.3).
Glyceraldehyde can alternatively be utilized by conversion to glycerol and
then to glycerol-1-phosphate. The latter is a substrate in the synthesis of
triacylglycerol, that is fat. Fructose and sucrose appear to promote obesity
42 4 Catabolism of sugars other than glucose

CH2OH
CH2OH CH2OH
O OH O O
OH 2 OH 2 OH
CH2OH CH2OH
OH O
OH OH
OH

Figure 4.2 Structures of fructose (left) and sucrose. In the sucrose molecule, the
fructose is “standing on its head”, and it is the carbon No. 2 that is joined to the
carbon No. 1 of glucose. Glucose is in its α configuration, whereas fructose is in its β
configuration.

more strongly than equivalent amounts of starch or glucose; if that is the case,
the utilization via glycerol-1-phosphate may be among the reasons.

4.1.1 Genetic defects in fructose assimilation


Fructose intolerance is a hereditary disease that is due to a homozygous defect
in the aldolase B gene. In this condition, fructose is still phosphorylated by fruc-
tokinase. The resulting fructose-1-phosphate, however, cannot be processed
further, and therefore the phosphate tied up in it cannot be reclaimed. Since
phosphate is required for the regeneration of ATP from ADP, this means that
ATP will be lacking, too, which will sooner or later destroy the cell. The disease
is characterized by potentially severe liver failure (Figure 4.4).
A situation resembling fructose intolerance was observed when fructose was
used as a substitute for glucose in intravenous nutrition of patients that for
some reason could not be fed orally. Application of large amounts of fructose
led to liver damage. Apparently, in these patients, aldolase B could not keep up
with fructose kinase, leading again to the accumulation of fructose-1-phosphate
and depletion of phosphate and ATP. Fructose therefore is no longer used as a
major component in intravenous nutrition.
A defect in the gene encoding fructokinase leads to a condition named fruc-
tosemia or fructosuria. As these names suggest, fructose levels are increased in
the blood1 and the urine. In this condition, no phosphate depletion occurs, and
the liver cells do not incur any damage. The condition is therefore not severe.

4.2 Metabolism of lactose and galactose


Lactose is the major carbohydrate contained in milk. It is a disaccharide of
glucose and galactose (Figure 4.5). Like maltose and sucrose, it is cleaved at
the brush border of the small intestine, and the monosaccharide fragments are
absorbed and passed along to the liver. The enzyme that accomplishes the
cleavage is lactase or, more precisely, β-galactosidase.
1 Haima = Greek for blood; hematology is the medical discipline dealing with diseases of the

blood.
4.2 Metabolism of lactose and galactose 43

O
Dihydroxyacetone−P
CH2OH H2C OH
O CH2OH ATP ADP OH P O
OH O O
O
OH CH2OH
O H2C C O P O
OH 1 H2
OH O
Fructose
OH
Fructose−1−P 2
OH

O H O H

HC OH O HC OH Fat
3 synthesis
C O P O C OH
H2 H2
O
Glyceraldehyde−3−P Glyceraldehyde

Figure 4.3 Degradation of fructose (fructolysis). 1: Fructokinase; 2: Aldolase B; 3:


Glyceraldehyde kinase.

After arriving in the liver, galactose is utilized by conversion to glucose (Fig-


ure 4.6a). It is first phosphorylated by galactokinase. The resulting galactose-
1-phosphate undergoes an exchange reaction with UDP-glucose, which is cat-
alyzed by galactose-1-phosphate uridyltransferase and releases glucose-1-phos-
phate and UDP-galactose. Glucose-1-phosphate can be converted by phospho-
glucomutase to glucose-6-phosphate (the first intermediate in glycolysis). UDP-
galactose is converted to UDP-glucose by UDP-galactose epimerase. This se-
quence of reactions constitutes a little metabolic cycle, in which UDP-glucose
and UDP-galactose fulfill a catalytic role but are not subject to any net turnover,
much like the intermediates in the citric acid cycle.

4.2.1 Genetic defects in lactose metabolism

A deficiency of the brush border enzyme lactase gives rise to a condition named
lactose intolerance, found frequently in people of East Asian descent past their
infant age. If lactose is not cleaved, it cannot be absorbed, so it makes its way
down the drain from the small into the large intestine. Many of the bacteria
found there have the capacity to metabolize lactose, which they will happily
convert to acids and gas. For example, Escherichia coli performs a mixed acid
fermentation. One of the products of this fermentation is formic acid (HCOOH),
which is then cleaved by formic acid lyase to H2 and CO2 .2 This leads to abdom-
inal discomfort and diarrhea. Since the environment in the large intestine lacks
2 The cleavage of formic acid serves the same purpose, namely detoxification of excess acid

derived from fermentation, as does ethanolic fermentation in yeast.


44 4 Catabolism of sugars other than glucose

Glyceraldehyde

Fructose Fructose-1-P × +
Dihydroxyacetone-P
ADP
ATP

1,3-Bis-P-glycerate

3-P-glycerate

Figure 4.4 Fructose intolerance. Due to a deficiency of aldolase B, fructose-1-


phosphate piles up inside the liver cells, and phosphate is depleted. ATP regeneration
from ADP stalls, which causes cell damage.

oxygen, H2 generated in the bacterial fermentation is not oxidized but instead


released as such, and in part is exhaled (Figure 4.7). An increase in exhaled
hydrogen gas upon ingestion of lactose can be used to diagnose the condition.
Treatment consists in omission of lactose in the diet. Milk can be pre-treated
with purified bacterial β-galactosidase, rendering it suitable for consumption
by lactose-intolerant individuals. Fermented milk products such as yoghurt
and cheese are depleted of lactose by microbial fermentation and therefore do
not pose a problem for lactose-intolerant individuals.
Other metabolic defects resemble those occurring in fructose metabolism.
Two different enzyme defects are known; somewhat confusingly, they are both
referred to as galactosemia, which means “galactose in the blood”.
1. A defect of galactokinase. In this case, galactose is simply not metab-
olized at all; it builds up in the blood and will mostly be eliminated in the
urine. The liver will not be adversely affected. However, there is a complication
elsewhere: Cataract (a cloudiness of the lens of the eye). This is believed to
occur by reduction of galactose to galactitol by aldose reductase (see below).
2. A defect of galactose-1-phosphate uridyltransferase. In this case, the sit-
uation resembles that outlined above for fructose intolerance: ATP is depleted
in the liver cells, because phosphate is trapped in galactose-1-phosphate, and
severe liver damage results.

4.3 The polyol pathway

Sorbitol is not strictly a sugar, since it lacks a keto or aldehyde group. It is


normally a minor component of dietary carbohydrates, but it is also prepared
synthetically and used as a sweetener. In addition, it is formed in our own
4.3 The polyol pathway 45

CH2OH
O
CH2OH CH2OH OH
OH O OH O O OH
OH OH OH

OH
OH OH

Figure 4.5 Structures of galactose (left) and lactose (Galactosyl-β-1,4-glucose).

metabolism from glucose (Figure 4.8). Degradation occurs via dehydrogenation


to fructose.
Aldose reductase can also reduce galactose, giving rise to galactitol. It is
believed that accumulation of sorbitol in diabetes melllitus and of galactitol in
galactosemia occurs in the lens of the eye and in peripheral nerves, and that
this contributes to the formation of cataract and to nerve damage in the two
diseases. Therefore, inhibitors of aldose reductase are being used—so far with
very limited clinical success—in the therapy of diabetes.
Formation of fructose from glucose via the polyol pathway occurs in the
seminal vesicles (part of the male sexual organs), and fructose is found in the
sperm. It serves as a supply of fuel to these cells in their quest of an oocyte;
the fructose is not utilized by the other tissues the sperm will get into contact
with.
Now, if that is the case, then inhibitors of aldose reductase should be great
as a pill for males, shouldn’t they? However, I have not seen any studies on
their effects on male fertility.
46 4 Catabolism of sugars other than glucose

O
a)
CH2OH HN
O
OH O O O N
CH2OH OH O P O P O C
OH O OH O
O O O
OH
O P OH
OH Glucose 1-P OH OH
O

ADP 2 O
1
ATP CH2OH HN

OH O
CH2OH
OH
O O O N
3
OH O
O P O P O C
OH O
OH
O O
OH
OH

OH OH

O
b)
CH2OH HN

OH O
O O O
OH N

O P O P O C
O
OH
CH2OH O O
O
O
O OH enzyme
OH OH
O P OR + NAD+
OH O
O

CH2OH
probable keto- HN
O
intermediate OH O O O N
OH O P O P O C
OH O
O O

OH OH

Figure 4.6 Metabolism of galactose. a: Pathway. 1: Galactokinase; 2: Galactose-


1-phosphate uridylyltransferase; 3: UDP-galactose epimerase. Not shown: Glucose-
1-phosphate can be converted to glucose-6-phosphate by phosphoglucomutase. b:
Mechanism of UDP-glucose epimerase. The requirement of this enzyme for NAD+
suggests that the epimerization of the hydroxyl group on C4 proceeds via reversible
abstraction of hydrogen from the substrate, that is via a keto intermediate.
4.3 The polyol pathway 47

Lactose Lactose
Liver

Galactose Galactose Small
+ + intestine
Glucose Glucose
Lung

bacterium

Liver
Large
CO2 intestine
H2

Figure 4.7 Lactose intolerance. Left: Normally, lactose is hydrolyzed by lactase, and
the constituent sugars absorbed in the small intestine. Right: In lactose intolerance,
lactase is missing, and lactose goes down the pipe into the large intestine, where
bacteria ferment it to hydrogen gas and other metabolites. Exhaled hydrogen can be
used as a diagnostic marker for lactose intolerance.

O OH OH
HC H2C H 2C

HC OH HC OH O
NADPH+H+ NADH+H+
HO CH HO CH HO CH

HC OH HC OH HC OH

HC OH HC OH HC OH
NADP+ NAD+
C OH C OH C OH
H2 H2 H2

Figure 4.8 The polyol pathway. Glucose is reduced to sorbitol by aldose reductase
(left); sorbitol is then dehydrogenated and turned into fructose.
Chapter 5

Pyruvate dehydrogenase and the citric


acid cycle

As discussed in the preceding chapters, degradation of one mole glucose to


pyruvate via anaerobic glycolysis only yields two moles of ATP. A much higher
yield can be obtained by subsequent complete, oxidative degradation of pyru-
vate to CO2 and H2 O, which is accomplished by the sequence of pyruvate de-
hydrogenase, the citric acid cycle, and the respiratory chain (Figure 5.1). These
transformations all occur in the mitochondria, while glycolysis occurs in the
cytosol. Therefore, before pyruvate can be completely degraded, it needs to
be transported from the cytosol to the mitochondrial matrix, across the two
mitochondrial membranes. While the outer mitochondrial membrane has non-
specific pores that allow free permeation of small metabolites, much like the
outer membrane of a gram-negative bacterium, the inner membrane is highly
selective and only allows passage of those metabolites for which specific carrier
systems exist. Pyruvate is shuttled into the mitochondrion by a specific carrier
system in exchange for hydroxide (OH – ).

5.1 Pyruvate dehydrogenase


Pyruvate dehdrogenase catalyzes the following overall reaction:

pyruvate + coenzyme A + NAD+ ------→


- acetyl-CoA + NADH + H+ + CO2 (5.1)

48
5.1 Pyruvate dehydrogenase 49

Glucose
Glycolysis
Cytosol
Pyruvate
Mitochondrial
transport

Pyruvate
CO2 Pyruvate
Mitochondrion dehydrogenase
Acetyl-CoA

Oxaloacetate Citrate
Citric acid cycle

Malate Isocitrate
H2 CO2
Fumarate α-Ketoglutarate
CO2
Succinate Succinyl-CoA

ADP + Pi Respiratory chain


O2
ATP

H 2O

Figure 5.1 The place of pyruvate dehydrogenase and the citric acid cycle in the
complete oxidative degradation of glucose. Glycolysis occurs in the cytosol, whereas
pyruvate dehydrogenase, the citric acid cycle, and the respiratory chain are located
in the mitochondria. A specific transport protein located in the inner mitochondrial
membrane is required to transport pyruvate to the mitochondrial matrix.

This reaction does not occur all at once but instead comprises a sequence
of group transfers and redox steps. We can distinguish five steps altogether,
which involve an equal number of coenzymes. The five reaction steps occur
at three different active sites, which are located on three different types of
enzyme subunits, which are bundled together into one large functional complex.
Pyruvate dehydrogenase therefore is a multi-enzyme complex. In each complex,
there are multiple copies of each of the three individual enzymes. The number
of subunits totals sixty in the E.coli version of pyruvate dehydrogenase (Figure
5.2) and is even higher in mammalian enzymes. Nevertheless, within this large
assembly, each subunit of any type is within easy reach of ones of the other
two types. This proximity ensures an easy flow of intermediate substrates from
one active site to the next during the sequential stages of the reaction, which
greatly increases the overall throughput. High throughput is a key advantage
of multi-enzyme complexes in general.
50 5 Pyruvate dehydrogenase and the citric acid cycle

a)

b)

TPP E1

S S
H
N

Lipoamide O
E2

FAD E3

Figure 5.2 Structure of the pyruvate dehydrogenase complex. a: Three-dimensional


model of the E.coli enzyme. The three different types of subunits are denoted as E1
(grey), E2 (white), and E3 (black). There are 24 subunits each of E1 and E2 , arranged
along the edges of a cube. The 12 subunits of E3 are placed on the planes of the cube. b:
For the mechanism of the reaction, all that matters is that the three different types of
subunits are within easy reach of each other, so that the substrates can be passed along
from one active site to the next. Coenzymes: Thiamine pyrophosphate (TPP) and Flavin
adenine dinucleotide (FAD) are bound to the active sites of E1 and E3 , respectively.
Lipoamide is bound to E2 , but its long, flexible arm allows it to interact with the active
sites of E1 and E3 as well.

The types of subunits are named according to the specific partial reactions
they catalyze. The first subunit is called pyruvate dehydrogenase, which name
therefore is ambiguous, denoting both the entire complex and a subunit. The
second subunit is called dihydrolipoyl transacetylase, and the third one dihy-
drolipoyl dehydrogenase. Instead of these explicit names, we will mostly use a
common shorthand notation and call them E1 , E2 and E3 in the following.
Figure 5.3 gives an overview of the sequential steps of the pyruvate dehy-
drogenase reaction:
1. Pyruvate is decarboxylated, and the remaining hydroxyethyl group be-
5.1 Pyruvate dehydrogenase 51

O O Figure 5.3 Reaction steps catalyzed


by the 3 subunits of pyruvate dehy-
O drogenase. E1 -E3 denote the three
different types of subunits; numbers
CH3
1-5 refer to the steps of the reac-
tion (see text). TPP: Thiamine py-
1 CO2 rophosphate; FAD: Flavin adenine din-
E1-TPP- ucleotide; CoA: Coenzyme A. E1 and
E3 each catalyze two steps, whereas
E1-TPP C OH
E2 catalyzes one.
CH3

2
CoA SH
HS
E2
H3C S
O
3
S

CoA S CH3 E2
S
O
HS
E2
HS

E3-FAD E3-FADH2

NADH + H+ NAD+

comes covalently bound to the TPP coenzyme at E1 .


2. The hydroxyethyl group is transferred to lipoamide, and is concomitantly
dehydrogenated to an acetyl group. This also occurs in the active site of E1 .
3. The acetyl group is transferred from lipoamide to coenzyme A. This
occurs in the active site of E2 .
4. Lipoamide is reoxidized, and the hydrogen is transferred to flavine ade-
nine dinucleotide (FAD) within the active site of E3 .
5. The reduced form of FAD—FADH2 —is reoxidized, and the hydrogen is
transferred to NAD+ . This step is catalyzed by E3 , too. NADH+H+ is then
released from the enzyme.
As you can see, each step in the pyruvate dehydrogenase reaction involves
52 5 Pyruvate dehydrogenase and the citric acid cycle

at least one coenzyme or cosubstrate.1 Therefore, this enzyme illustrates quite


nicely the great importance of coenzymes and cosubstrates in enzyme catalysis.

5.2 Catalytic mechanisms in the pyruvate dehydrogenase


reaction
The function of the first coenzyme—thiamine pyrophosphate (Figure 5.4a)—
consists in providing a carbanion (Figure 5.4b), by which we mean a negative
charge on a carbon atom. Carbanions are very powerful nucleophiles, and
the TPP carbanion functions as such in the decarboxylation of pyruvate. It
attacks the keto group of pyruvate, which leads to a covalent intermediate from
which CO2 is cleaved. This yields a second carbanion, now located within the
hydroxyethyl group that is the remainder of the substrate. The new carbanion
cleaves the disulfide of lipoamide, upon which the entire substrate is cleaved
from TPP and carried away by lipoamide (Figure 5.4c).
As pointed out above (see Figure 5.2b), lipoamide is covalently attached to
E2 but is able to access the active sites of all three types of subunits. Its next
destination is E2 , where transfer of the acetyl group to coenzyme A occurs.
This reaction is straightforward, as it consists simply in the formation of one
thioester at the expense of another. It proceeds once more by nucleophilic
attack of a thiolate anion on a carbonyl group (Figure 5.4).
After the transacetylation from lipoamide to coenzyme A is completed,
the only remaining task is to restore lipoamide to its disulfide form. This is
accomplished by E3 , which transfers the hydrogen first to FAD (which is tightly
bound to the enzyme) and from there to NAD+ . The structure of FAD in its
oxidized and reduced forms is shown in Figure 6.5.

5.3 Regulation of pyruvate dehydrogenase


Pyruvate is used in various pathways:
1. It can be turned into acetyl-CoA for complete degradation and for fatty
acid or cholesterol synthesis,
2. it can be carboxylated to yield oxaloacetate, to be used either in gluconeo-
genesis or in the citric acid cycle, and
3. it can be used for the synthesis of amino acids. For example, a single
transamination reaction turns pyruvate into alanine.
It is clear then that the activities of all enzymes that act on pyruvate, includ-
ing pyruvate dehydrogenase, have to be regulated in keeping with the prevailing
metabolic needs. Pyruvate dehydrogenase is subject to two modes of regulation
(Figure 5.6):
1 The last step provides an example of the distinction between coenzymes and cosubstrates:

FAD is a coenzyme, since it is in the same state before and after the reaction. NADH is a
cosubstrate, since it undergoes a net turnover, just like the substrate does.
5.3 Regulation of pyruvate dehydrogenase 53

CH3
a) O O b)
+ O P O P OH
N N
O O
S
H3C N NH2 O

CH3
O
H
+ R
N N

S
c) CH3 CH3 H3C N N H H
H
R2 + R1 R2 + R1
N N

C S S
O O
CH3 C CH3
O O O H
O O
+
H
CH3
O
CH3 CH3
H + R
CO2 N N
R2 + R1 R2 R1
N N S
H3C N N H
S O S
H +
H
S C CH3 C C CH3
Lip O
O H O H
S
+
H

CH3 CH3
O
R2 + R1 R2 + R1
N N CH3
OH
S C S
+ R
N N
S C CH3 S C CH3
Lip Lip C S
O H O + H3C N NH
SH SH H H

Figure 5.4 Catalytic mechanism of E1 . a: Structure of thiamine pyrophosphate (TPP);


b: Electron pushing scheme to account for the formation of the carbanion (C – , blue); c:
Mechanisms of decarboxylation of pyruvate, and of transfer of the hydroxyethyl group
onto lipoamide.

1. Allosteric control (see section 2.5). Pyruvate dehydrogenase is stimulated


by Fructose-1,6-bisphosphate but inhibited by NADH and Acetyl-CoA.
2. Phosphorylation by a special regulatory enzyme, pyruvate dehydrogenase
kinase. This enzyme is tightly bound to the pyruvate dehydrogenase complex.
Phosphorylation inactivates pyruvate dehydrogenase. The kinase, in turn, is
allosterically activated by NADH and Acetyl-CoA but inhibited by ADP, NAD+
and by free coenzyme A.
3. Phosphorylation is reversed, and the activity of pyruvate dehydrogenase
restored by a phosphatase, which is also associated with the pyruvate dehydro-
54 5 Pyruvate dehydrogenase and the citric acid cycle

a) CH3 H
O b) CoA S H
+

C C C C N C C
H
O NH H3C C S
CH3 OH O
O P O C O Lip
O
O NH2 C S C CH3 HS

O P O N
N
O CoA S H
+

C N
O N
H3C C S

O Lip
HS
O OH
O P O
CoA S
O

H3C C HS

O Lip
HS

Figure 5.5 The dihydrolipoyl-transacetylase (E2 ) component of PDH transfers the


acetyl group from dihydrolipoamide to acetyl-CoA. a: Structure of acetyl-CoA. Only a
small part—the sulfur atom at the very tip of the molecule—directly engages in the
acetyl group transfer. b: Reaction mechanism. One thiol ester bond gives way to
another by way of nucleophilic attack.

PDH-P (inactive)
Ca++

Pyruvate +
- NAD+ Phosphatase
Kinase
CoA-SH
+

PDH (active)
- +
Fructose-1,6-bis-P
NADH
Acetyl-CoA

Figure 5.6 Regulation of pyruvate dehydrogenase. Plus signs indicate allosteric acti-
vation, minus signs allosteric inhibition. PDH-P: Phosphorylated pyruvate dehydroge-
nase.
5.4 The citric acid cycle 55

genase complex.
All of the above regulatory effects make good physiological sense. NADH
and acetyl-CoA inhibit PDH, which means that the enzyme will slow down when
its products accumulate. Such feedback inhibition is a straightforward way to
link the activity of a pathway to the metabolic requirements it serves. On the
other hand, pyruvate, NAD+ and fructose-1,6-bisphosphate apply feed-forward
activation – as more substrate arrives, the enzyme should pick up speed.
One more interesting detail is that the PDH phosphatase is activated by
calcium ions. Calcium ions are also involved in triggering the contraction of
muscle cells; activation of PDH will bolster the replenishment of ATP that is
consumed in the contraction.

5.4 The citric acid cycle


With the conversion of pyruvate to acetyl-CoA, the carbon derived from glucose
has reached a central hub of energy metabolism. Degradation of all foodstuffs—
carbohydrates, amino acids, and fat—proceeds through this stage. The next
step toward complete oxidation is the citric acid cycle, or tricarboxylic acid
cycle (TCA). Its basic idea consists in releasing the carbon as CO2 , and retaining
the hydrogen for “cold combustion” in the respiratory chain. However, if we
look more closely, we see that something is missing from this description. For
the sake of simplicity, let us get rid of the coenzyme A by converting acetyl-CoA
to acetate:2
CH3 CO-S-CoA +H2 O ------→
- CH3 COOH + CoA-SH (5.2)

If we look back at figure Figure 5.1, we see that the TCA produces 4 molecules
of H2 and two molecules of CO2 . Now, if we attempt to balance the acetate with
these amounts of CO2 and H2 :

CH3 COOH ------→


- 2 CO2 + 4 H2 (5.3)

we see that we are short 4 hydrogens and 2 oxygens on the left side. However,
we can balance the equation if we add two molecules of water:

CH3 COOH + 2 H2 O ------→


- 2 CO2 + 4 H2 (5.4)

Therefore, half of the hydrogen produced in the TCA is gained by the reduction
of water. The water-derived oxygen is used to complete the oxidation of the
acetyl carbon. Hydrogen derived both from water and the acetyl group is then
re-oxidized in the respiratory chain to generate ATP.
The energy yield of the TCA itself, in terms of directly generated energy-rich
phosphoanhydride bonds, is very modest – just one molecule of GTP, which
2 The hydrolysis of coenzyme A actually takes place at the stage of citryl-CoA, not acetyl-CoA;

however, this makes no difference to the overall balance of the TCA.


56 5 Pyruvate dehydrogenase and the citric acid cycle

Figure 5.7 The citrate syn-


thase reaction. Abstrac- CoA CoA
tion of a proton by a basic S S
residue in the active site trig-
C O C O
gers nucleophilic attack by BH2
BH
+
H3
acetyl-CoA on oxaloacetate. H3C H2C
The reaction is pulled for-
ward by the subsequent hy- HOOC C C COOH HOOC C C COOH
H2 H2
drolysis of CoA. O
O
+
H

CoA
H2O CoA
SH
O S

+
C O +
C O
BH
H3 BH
H3
CH2 CH2

HOOC C C COOH HOOC C C COOH


H2 H2
OH OH

is equivalent to ATP, is generated for each molecule of acetyl-CoA degraded,


compared to approximately 15 in the respiratory chain. It is clear therefore
that the TCA’s main contribution to ATP generation is to provide H2 for the
respiratory chain.

5.5 Reactions in the citric acid cycle


The individual reactions in the TCA are outlined in Figures 5.7 and 5.8:
1. The acetyl group of acetyl-CoA is added to the carbonyl group of ox-
aloacetate (Figure 5.7). Coenzyme A is released in the process. This reaction
is carried out by citrate synthase. The key aspect of the mechanism is the
abstraction of a proton from the acetyl group, which creates a carbanion that in
turn reacts with the carbonyl bond of oxaloacetate to create a hydroxyl group.
2. The newly formed hydroxyl group is shifted to an adjacent carbon (Figure
5.8a) to yield isocitrate. The latter reaction is catalyzed by citrate isomerase
and involves the transient abstraction of water across the two carbons involved;
the water is then added back in the reverse orientation.
3. Isocitrate is decarboxylated and dehydrogenated by isocitrate dehydroge-
nase, which yields α-ketoglutarate. In contrast to the pyruvate dehydrogenase
reaction, dehydrogenation precedes decarboxylation. This is shown by the oc-
currence of the intermediate depicted in Figure 5.8b, known as oxalosuccinate.
5.5 Reactions in the citric acid cycle 57

H2O H2O
a) H2C COOH H2C COOH H2C COOH

HO COOH COOH HC COOH

H2C COOH HC COOH HO CH COOH

b) NAD NADH+H+ CO2


H2C COOH H2C COOH H2C COOH

HC COOH HC COOH CH2

HO CH COOH O C COOH O C COOH

c) CO2 NADH+H+
H2C COOH H2C COOH
NAD+
CH2 CH2

O C COOH O C S CoA
Coenzyme A -SH

d)
H2C COOH H2C COOH H2C COOH

CH2 CH2 O CH2

O C S CoA O C O P O O C O

O
Phosphate GDP GTP

e)
NADH+H+
NAD+
COOH COOH COOH COOH
FADH2
CH2
FAD HC HO CH C O

CH2 CH CH2 CH2

COOH 1 COOH
2 COOH 3 COOH
H2 O

Figure 5.8 Further reactions in the citric acid cycle. a: The citrate isomerase reaction
proceeds via a dehydrated intermediate (called cis-aconitate). b: Isocitrate dehydro-
genase transfers hydrogen to NAD+ to generate oxalosuccinate as an intermediate,
which it then decarboxylates. c: The α-ketoglutarate dehydrogenase reaction is anal-
ogous to the one catalyzed by pyruvate dehydrogenase. d: The succinate thiokinase
reaction utilizes the energy-rich thioester bond of succinyl-CoA to generate GTP. The
reaction proceeds via a succinylphosphate intermediate. e: Succinate dehydrogenase
(1), fumarase (2), and malate dehydrogenase (3) regenerate oxaloacetate to close the
cycle.
58 5 Pyruvate dehydrogenase and the citric acid cycle

There are two isozymes of isocitrate dehydrogenase; one uses NAD+ and the
other NADP+ as the cosubstrate. The role of these two isozymes is considered
in section 6.6.
4. α-Ketoglutarate is converted to succinyl-CoA by α-ketoglutarate dehy-
drogenase (Figure 5.8c). This step is completely analogous to the pyruvate
dehydrogenase reaction. The analogy is reflected in a high degree of homology
between the subunits of the two enzymes. If you look closely at the mecha-
nism (Figure 5.3, above), you will see that the reactions carried out by the final
subunit (E3 ) will be identical in both cases, since E3 interacts with lipoamide
when only hydrogen is left but the rest of the substrate is gone. Indeed, the two
enzyme complexes share the very same E3 protein; only E1 and E2 are specific
for the respective substrates. The same E3 occurs yet again in an analogous
enzyme that participates in the degradation of the branched chain amino acids
(section 12.5.2).
5. Succinyl-CoA is converted to succinate by succinate thiokinase, and GDP
is concomitantly phosphorylated to GTP. We know from the glyceraldehyde-3-
phosphate dehydrogenase reaction (Figure 3.6) that thioester bonds are energy-
rich and can drive the phosphorylation of carboxylic acids.3 A carboxylic acid
phosphate, succinylphosphate, also occurs as an intermediate in the succinate
thiokinase reaction (Figure 5.8d). As with phosphoglycerate kinase, the phos-
phate group is then transferred to a nucleotide diphosphate. This nucleotide
is GDP instead of ADP; however, the energy content of the phosphoanhydride
bonds created in GTP and ATP is virtually the same.
6. Succinate is dehydrogenated across the CH–CH bond by succinate de-
hydrogenase to yield fumarate (Figure 5.8e, left). The coenzyme used in this
reaction is flavin adenine dinucleotide (FAD). As a rule of thumb, you can as-
sume that FAD is used in the dehydrogenation of CH–CH bonds, whereas either
NAD+ or NADP+ are used in the dehydrogenation of CH–OH bonds. While all
other enzymes in the TCA are in aqueous solution in the mitochondrial matrix,
succinate dehydrogenase is bound to the inner surface of the inner mitochon-
drial membrane; it is identical with complex II of the respiratory chain (see
section 6.1).
7. Fumarate is hydrated to L-malate by fumarase (Figure 5.8e, middle).
8. Malate is dehydrogenated by malate dehydrogenase to yield oxaloacetate
(Figure 5.8e, right).
The malate dehydrogenase reaction regenerates oxaloacetate, and thus
closes the cycle. It is noteworthy that its equilibrium favours malate, so that
the concentration of oxaloacetate is very low. This has two consequences:
1. The availability of oxaloacetate is a kinetic bottle neck that controls the
rate of the initial reaction of the TCA, that is the synthesis of citrate.
3 With glyceraldehyde-3-phosphate dehydrogenase, the reaction proceeds in the opposite direc-

tion in glycolysis, but it is reversible and functions in the opposite reaction in gluconeogenesis.
5.6 Regulation of the citric acid cycle 59

2. The low concentration of oxaloacetate also detracts from the free energy
that is released by the citrate synthase reaction. To make that reaction go
forward, it is necessary to sacrifice the energy-rich bond of the CoA thioester,
which in contrast to the succinate thiokinase reaction is simply hydrolyzed and
not utilized for the generation of a molecule of GTP or ATP.

5.6 Regulation of the citric acid cycle


Acetyl-CoA is not only utilized for complete oxidation but also for biosynthetis,
most notably of fatty acids and of cholesterol. Therefore, the activity of the cit-
ric acid cycle must be regulated in keeping with the prevailing metabolic needs.
Major regulatory factors are: (1) The concentration of oxaloacetate, which limits
the rate of the citrate synthase reaction. (2) Inhibition of isocitrate dehydroge-
nase and of α-ketoglutarate dehydrogenase by NADH, the major direct product
of the TCA, and by ATP, the ultimate product of complete oxidation via the
TCA and the respiratory chain.
Intriguingly, the inhibition by NADH and ATP applies only to the NAD+ -
dependent isocitrate dehydrogenase, but not to the NADP+ -dependent one,
which in cells with high TCA activity such as heart and skeletal muscle cells has
a much higher activity than the former. How, then, is this enzyme regulated?
This appears to occur in coordination with the flow through the respiratory
chain and the proton-motive at the inner mitochondrial membrane. The mech-
anism is quite fascinating and is discussed at the end of the following chapter.
Chapter 6

The respiratory chain

In the respiratory chain, the NADH and FADH2 accumulated in the preceding
degradative pathways, mostly the TCA, is finally disposed of by reacting it
with molecular oxygen. The free energy of this “cold combustion” is used
to generate ATP. While a small amount of ATP (or GTP) is produced in those
preceding pathways, the contribution of the respiratory chain is the largest
by far. This is the reason that only aerobic metabolism allows us to sustain
physical exertion for extended periods of time.
The workings of the respiratory chain are quite different from all other
pathways in human metablism (see Figure 6.1). Those other pathways consist of
a succession of discrete enzymatic reactions. In all the ATP-producing reactions
that we have seen so far, the energy was always passed from one energy-rich
bond to the next. In contrast, the respiratory chain combines chemical reactions
with physical forces that are not pinned down to individual molecules, and the
energy is stored and converted in novel ways. The entire process consists of
the following stages:
1. Abstraction of the hydrogen from NADH and FADH2 , and separation of
the hydrogen into protons and electrons.
2. Passage of the electrons down the electron transport chain, which is a
cascade of redox cofactors that are bound to four large protein complexes em-
bedded in the inner mitochondrial membrane. The protein complexes function
as proton pumps; migration of the electrons along this chain makes them expel
protons from the mitochondrion. For each electron migrating down the chain,
multiple protons are pumped out of the mitochondrion.

60
6.1 ATP synthesis can be separated from electron transport 61

Cytosol
H+ H+ H+ H+ H+ H+
+ +
H H
C
I
Q ATP synthase
II III IV
H+ e- e- H+ H+ ADP + P

NADH FADH2 ATP


+ + - O2
H H e

Mitochondrial matrix
H2O H+

Figure 6.1 Overview of the respiratory chain. Hydrogen provided by NAD+ or FAD is
split into protons and electrons. The electrons are passed along a cascade of proteins
located in the inner mitochondrial membrane, complexes I–IV, that use the energy
provided by the electron flow to pump protons out of the mitochondrial matrix, into
the cytosol. The electrons then react with oxygen and are rejoined by protons to yield
water. The protons accumulated outside the mitochondrion flow back in through ATP
synthase, which is thereby induced to rotate. The rotary motion is then harnessed to
drive the synthesis of ATP from ADP and ionic phosphate. Q: Ubiquinone (coenzyme
Q); C: Cytochrome C. Note that the reactions as shown here are not stoichiometrically
balanced.

3. At the end of the electron transport chain, the electrons are scooped up
by oxygen, which then combines with protons to yield water.

4. The protons accumulated outside the mitochondrion are allowed back in


through another membrane protein, ATP synthase. This protein is a molecular
motor, driven to rotate by the flow of protons into the cytosol. The rotary
motion of ATP synthase in turn drives the synthesis of ATP from ADP and
phosphate.

As mentioned before, the outer mitochondrial membrane is permeable for


most small molecules and ions, and therefore the proton concentration between
the two mitochondrial membranes is in equilibrium with the cytosol. The
proton concentration gradient therefore exists across the inner mitochondrial
membrane only.

If you think that all this sounds rather strange and vague, you are right –
but don’t let that trouble you. The purpose here is only to divide and conquer,
that is to break up the overall process into manageable parts, which we can
then tackle in more detail in their turn.
62 6 The respiratory chain

a)
O O
H+ NO2 NO2

H+
H+
NO2 NO2
H+
H+
H+
OH
OH OH
OH H+
NO2 NO2

H+

NO2 NO2

b)
H+ H+ H+
C
I
Q
II III IV
- e-
+e
H H+ H+
Figure 6.2 Uncoupling of the respiratory chain. a: The decoupling compound dini-
trophenol can diffuse across the inner mitochondrial membrane in both its protonated
and unprotonated form. It can therefore carry protons into the mitochondrion, thereby
dissipating the driving force for the ATP synthase. In the presence of dinitrophenol,
electron transport and hydrogen oxidation (molecular respiration) will continue, but
ATP synthesis will cease. b: The same thing happens with uncoupling proteins.

6.1 ATP synthesis can be separated from electron trans-


port
The first thing to note about electron transport and ATP synthesis is that they
can be experimentally separated from each other. Electron transport occurs
without ATP synthesis in the presence of uncoupling agents. These are proton-
carrying compounds such as dinitrophenol, which facilitate the transport of
protons downhill their concentration gradient, that is back into the mitochon-
drion.1 This will short-circuit the proton gradient and prevent the ATP synthase
1 In general, charged compounds do not cross membranes efficiently. However, dinitrophenol

can cross the mitochondrial membrane not only in protonated, neutral but also in the deproto-
nated, charged form. This is because the charge in dinitrophenol is highly delocalized, that is
spread across the enrtire molecule.
6.1 ATP synthesis can be separated from electron transport 63

Light H+
H+ H+ ADP+Pi
H+
H+ H+
H+ H+
H+ ATP
H+

Figure 6.3 The Racker experiment. Bacteriorhodopsin is a light-driven proton pump.


A vesicle containing both bacteriorhodopsin and ATP synthase will accumulate protons,
which will drive ATP synthase. Note that the orientation of both proteins is inside out
relative to that found in the natural (mitochondrial or bacterial) membranes.

from functioning (Figure 6.2a). The same functional effect is brought about by
uncoupling proteins (Figure 6.2b). These are passive proton transporters, lo-
cated in the inner mitochondrial membrane and found in particularly high
concentration in a special tissue called brown fat. Their physiological signifi-
cance is discussed in section 10.3.1.
The effect of uncouplers shows that electron transport can occur in the
absence of ATP synthesis. On the other hand, ATP synthesis will occur in the
absence of electron transport if another means is provided to sustain a proton
gradient. An experimental system, devised by Ephraim Racker, is shown in
Figure 6.3. Here, a molecule of ATP synthase has been incorporated inside
out into a liposome, that is a small artificial membrane particle, along with a
molecule of bacteriorhodopsin. The latter is a quite remarkable protein, found
in a certain bacterium (Halobacterium halobium), that functions as a light-
driven proton pump. Accumulation of protons inside the liposome will set the
ATP synthase in motion and drive the synthesis of ATP.
The significance of these findings is that, despite their presence in the same
membrane and their close proximity, the proton gradient is the only functional
link between the electron transport chain and ATP synthase: The electron
transport chain generates the proton gradient, whereas ATP synthase puts it to
work and thereby dissipates it. Because of this clear separation, we can safely
examine these two functions separately from each other.
64
complex I mitochondrial matrix
complex II
complex III

complex IV

TM domain

6 The respiratory chain


cytoplasmic side

Figure 6.4 Structures of the four respiratory chain complexes that form the respiratory chain. The structure of Complex I
is only known in part, and a lame outline has been substituted for the missing trans-membrane domain. Redox cofactors are
highlighted. Yellow/grey blobs represent iron sulfur clusters. Organic rings (green) with grey balls (iron atoms) in the center
are hemes; other rings are flavins or ubiquinone. Drawn with pymol from pdb files 3ias, 2fbw, 3cx5 and 2zxw, after a figure in
Biochemistry 42:2266–2274 (2003).
6.2 The electron transport chain 65

6.2 The electron transport chain


Figure 6.4 shows structural sketches of the four protein complexes that form
the respiratory chain. Each of the four complexes has a specific role in the
electron transport process:
1. Complex I accepts hydrogen from NADH + H+ and is therefore also called
NADH dehydrogenase. The NADH is oxidized back to NAD+ and thereby read-
ied for the next round of reduction in the TCA or by pyruvate dehydrogenase.
The two hydrogen protons are expelled, as are apparently two additional pro-
tons. The electrons are thereafter transferred to the small carrier molecule
ubiquinone (see Figure 6.5c).
2. Complex II accepts hydrogen from succinate. It was mentioned before
(section 5.5) that it is identical with succinate dehydrogenase, which illustrates
that the two pathways are really functionally one. The electrons are again
transferred to coenzyme Q, but no proton extrusion occurs at complex II.
3. Complex III reoxidizes coenzyme Q and expels protons. According to
the coenzyme Q cycle model presented below, four protons are being expelled
at this stage for each pair of electrons transported, but in some sources the
number of protons are expelled is given as two. This example shows that there
is still some uncertainty about the mechanistic details of the electron transport
chain. The electrons are being delivered to the small electron carrier protein
cytochrome C.
4. Complex IV reoxides cytochrome C and is therefore also called cytochrome
C oxidase. The electrons are transferred to oxygen, and the considerable free
energy associated with this electron transfer step is utilized to expel up to 4
protons from the mitochondrial matrix.

6.2.1 Redox cofactors in the electron transport chain

Electrons do not occur in free form but are always part of molecules or ions2
Therefore, to make electrons flow along the prescribed path along complexes
I-IV, these proteins must provide functional groups that are able of accepting
and donating electrons. These groups must be closely spaced, within a few
Angstroms of each other, to allow for efficient electron transfer. Furthermore,
to persuade the electrons to flow in the right direction, the successive transi-
tions must be exergonic, that is their free energy (∆G) must be negative.
In Figure 6.4, you can see a multitude of redox cofactors, neatly spaced along
the protein molecules, that function as “stepping stones” for the migrating
electrons. These prosthetic groups fall into five classes:
2 Electrons do occur free as β-particles in ionizing β-radiation. However, to escape capture,

β-particles must possess an amount of energy much higher than those available in biochemical
or other chemical reactions. This high energy causes them to break up any molecules in their
path into ions or radicals.
66 6 The respiratory chain

O
a)
O P O R

OH O
R = H: FMN
OH R = AMP: FAD
OH

OH
H
H3C N N O
H3C N N O

NH
NH
H3C N
H3C N H

b) R R c)
O
R R
N N H3C CH3O

N N H
R R CH3O

CH3 O
n
R R

d)
Cys Cys Cys
Cys

Cys Cys

Cys Cys
Cys
Cys Cys Cys

Figure 6.5 Structural families of redox cofactors in the respiratory chain. a: Flavine
nucleotides (FAD and FMN), oxidized (left) and reduced (right). b: A heme cofactor with
an iron atom coordinated by the four nitrogen atoms of the ring. The nature of the ‘R’
residues differs in the various heme cofactors, which will contribute to their distinct
redox potentials. c: Ubiquinone. The isoprenyl tail is very long (n=10) and hydrophobic,
so that ubiquinone is confined to the membrane interior. See Figure 6.8 for details on
the redox chemistry. d: Iron sulfur clusters. 1, 2 or 4 of the sulfurs coordinated to
each iron atom can be part of cysteine side chains; the others will be free sulfide.
6.2 The electron transport chain 67

1. Flavins (Figure 6.5a). We have already encountered flavin adenine dinu-


cleotide (FAD) as the redox coenzyme used by succinate dehydrogenase. Both
FAD and the related flavin mononucleotide (FMN) occur in the respiratory chain.
2. Porphyrins or hemes (Figure 6.5b). These are tetrapyrrol rings that hold
a central iron ion which can adopt different oxidation states (mostly Fe2+ and
Fe3+ , although Fe4+ occurs within complex IV). The name “cytochrome” is in
some cases applied to the hemes themselves, in others to the entire complex
of the heme and the protein it is bound to.
3. Iron-sulfur clusters. Here, it is again the iron that accepts and donates
electrons by alternating between different oxidation states. Each iron ion is held
in place by four sulfur atoms. These can be cysteine sulfurs or free sulfides
(S2- ) in various proportions, which gives rise to different sizes of iron-sulfur
clusters (Figure 6.5c).
4. Cytochrome C oxidase (complex IV) contains copper ions that function as
redox cofactors in the final transfer of electrons to oxygen. These copper ions
are coordinated—that is, bound—directly by amino acid side chains, without
any additional prosthetic group.
Since there are many more than four individual cofactors in the respira-
tory chain, most classes occur in multiple instances. Importantly, the redox
potentials (see below) of the different cofactors that belong to the same chem-
ical class can be different due to influences from their respective molecular
environments.
In addition to the stationary redox cofactors that occur within complexes
I-IV, there are two electron carriers that are not tighly associated with one
individual complex but function as shuttles between them:
1. Ubiquinone or coenzyme Q (Figure 6.5c). This coenzyme contains a
quinone group. It carries electrons, as hydrogen, from complexes I and II
respectively to complex III. It also contains a long hydrophobic poly-isoprene
tail, which confines it to the hydrophobic interior of the membrane. Its special
mode of reoxidation is considered in some detail below (Figure rchainQcycle).
2. Cytochrome C. This is a small protein that again contains a heme. It is
located at the outer surface of the inner mitochondrial membrane and shuttles
electrons between complex III and complex IV.

6.2.2 Electrochemistry of the respiratory chain

It was stated above that the free energy of the transfer of an electron from one
cofactor to the next must be negative in order to make it happen. Another way
of saying the same thing is that the cofactors should have progressively higher
redox potentials. The reason why redox potentials are commonly used in this
context is that they can be measured more directly than ∆G. However, free
68 6 The respiratory chain

0 0
a) ∆E0 > 0 b) ∆E0 < 0

e–
e– e–
e– e–
e– e– 2 e– 2 e–
1
2 H2 H+ Q Q+ 2 H+ H2 NADH NAD+
+ H+

Figure 6.6 Experimental setup for measuring standard potentials. The sample and
the reference are contained in two adjacent chambers. Platinum electrodes are im-
mersed in the solutions. Electrons are withdrawn from one chamber and delivered into
the other, flowing through a voltmeter indication the direction and magnitude of the
potential difference. Protons and other ions can flow across a salt bridge to maintain
electroneutrality. Left: Coenzyme Q withdraws electrons from the standard hydrogen
0
electrode and accordingly has a positive ∆E0 . Right: NADH feeds electrons into the
0
standard electrode, making its ∆E0 positive.

energy and redox potential are directly related by the following equation:

∆G = −∆E n F (6.1)

where ∆E is the difference in the redox potentials between two cofactors, F


is Faraday’s constant: 96,500 Coulombs/mol, and n is the number of electrons
transferred.3 For example, NADH feeds two electrons at a time into the chain,
so the value of n for this reaction is 2.
One can look at the redox potential of a cofactor as its affinity for electrons
– the higher it is, the more strongly the cofactor will attract electrons.4 The
redox potential is a useful parameter since it can readily be measured. Figure
6.6 shows the required experimental setup:
1. A chemically inert electrode, often platinum, is immersed in a solution
that contains standard amounts of the reduced and oxidized forms of our
carrier,

3 Another thing to remember is that the unit of ∆E, Volt, is defined as J/Coulomb, since voltage
= energy/charge.
The minus sign in this equation results from the fact that the electron-donating electrode, the
cathode, is considered negative by convention. It is fundamentally meaningless but handy as a
trap in exam questions.
Faradays constant can be considered a historic artifact, resulting from the fact that units of
electricity were already defined before the principles of electrochemistry were understood.
4 You have encountered the same concept with chemical elements as their electronegativity:

An element with a high electronegativity holds on to electrons particularly firmly, that is it has a
high affinity for electrons.
6.2 The electron transport chain 69

2. a reference electrode is immersed in a bath of an electron carrier of known


redox potential, and
3. a voltmeter connects the two compartments.
Another connection must be made between the two baths so as to permit flow
of ions to preserve electroneutrality but prevent mixing of the contents by
convection. This may be a small hole plugged with agar.
The standard reference electrode commonly used in chemistry is the plat-
inum/hydrogen electrode. This electrode contains hydrogen both in reduced
form—H2 , kept constant by equilibration of the aqueous phase with hydrogen
gas at a pressure of 1 atmosphere—and in the oxidized form, H+ , which is
adjusted to 1 mol/l or pH 0. Platinum functions both as a conductor and as a
catalyst for the interconversion of reduced and oxidized hydrogen.
The potential of a redox carrier measured against this electrode is defined
as its standard redox potential or ∆E0 . For biochemical purposes, a modified
standard electrode is used, which has a pH of 7 instead of 0, and the redox
0
potentials measured against this electrode are referred to as ∆E0 . A pH of 7 is
just as arbitrary a choice as pH 0, but we will stick with it because the books
do so, too.
The redox potentials of several selected carriers in the respiratory chain are
shown in Figure 6.7. The lowest value is found with NAD+ , in keeping with
its position at the start of the transport chain. The next carrier in sequence,
FMN, is part of complex I. It has a slightly higher potential than NADH and is
therefore able to abstract its electrons.
The iron-sulfur cluster N2, which occupies the lowermost position within
complex I as shown in Figure 6.4, has a significantly higher potential than the
FMN. This step in potential corresponds to a significant amount of free energy
that is released at some point between FMN and N2. Complex I uses this energy
to expel protons from the mitochondrion, against their concentration gradient.
Major steps in potential that drive proton expulsion also occur within complex
III and complex IV.
Only minor steps of potential occur in all other stages, including the delivery
of electrons from complex I and complex II—the latter is represented in the
figure by FADH2 —to complex III via ubiquinone, and between complexes III
and IV via cytochrome C. These minor steps suffice to drive electron transport
forward, but they are too small to drive any additional external work (see
below).
The redox potential increases continuously along the respiratory chain to
reach its highest value at oxygen, which therefore has the highest affinity for
the electrons and gets to keep them. Reduced oxygen, which recombines with
protons to yield water, therefore is the end product of respiration.
While the scale of redox potentials establishes the general direction of elec-
tron flow, the redox cofactors also differ in two other important aspects:
70 6 The respiratory chain

−0.4 NADH 75.2


FMN

−0.2 complex I 36.6


(Fe-S)N-2 FADH2 (complex II)

∆G (KJ/2 mol electrons)


0 CoQ −2.0

complex III
Heme c1 CytC
∆E0 (V)

0.2 −40.6
Heme a3
0

0.4 −79.2

0.6 complex IV −117.8

O2
0.8 −156.4

1.0 −195.0

Figure 6.7 Redox potentials for selected cofactors along the respiratory chain. Elec-
trons are fed either from NADH into complex I or from FADH2 into complex II. The
scale on right hand side shows the conversion of ∆E to the molar ∆G, per pair of elec-
trons (see equation 6.1). As ∆E goes up, ∆G goes down, driving the electron transport
forward. Major jumps in potential occur within complexes I, III and IV; these are the
sites that harness the free energy of electron transport to proton export. Abbreviations:
CoQ, coenzyme Q (ubiquinone); CytC, cytochrome C; Fe-S, iron sulfur cluster.

1. NADH, FADH2 , FMNH2 and coenzyme Q carry both electrons and protons
– that is, hydrogen. In contrast, the hemes and the iron-sulfur clusters carry
only electrons.
2. NAD+ can only accept and donate pairs of electrons, whereas the hemes
and iron-sulfur clusters can only accept and donate single electrons.
The switch from the two-electron carrier NADH to the one-electron carrying
Fe−S clusters within complex I is mediated by FMN, which can accept or donate
electrons both pairwise and singly:

- NAD+ + FMNH2
NADH + H+ + FMN ------→ (6.2)
III · + II
FMNH2 + Fe −S ------→
- FMNH + H + Fe −S (6.3)
· III + II
FMNH + Fe −S ------→
- FMN + H + Fe −S (6.4)

After accepting H2 from NADH + H+ (equation 6.2), FMNH2 donates the


electrons one by one to the first Fe−S cluster (equations 6.3 and 6.4), adopting
a sufficiently stable radical form between these two transfers.
The electron transfer between FMNH2 and Fe−S also illustrates what hap-
pens if an electron-only carrier is reduced by a hydrogen carrier: The protons
are simply shed into the solution. Conversely, when a hydrogen carrier is re-
duced by an electron carrier, as happens with the reduction of ubiquinone by
6.2 The electron transport chain 71

(Fe–S)N2, protons are taken up from the solution:

Fe II −S + H+ + Q ------→
- QH + Fe III −S
·
(6.5)
Fe II −S + H+ + QH ------→
·
- QH2 + Fe III −S (6.6)

Hydrogen carriers alternate with electron-only carriers at several points in


the chain. This means that electrons are stripped of their protons and rejoined
by protons again repeatedly during transport. Where protons are stripped
off, they are preferentially released at the cytosolic side, whereas new protons
are taken up from the mitochondrial side. This accounts for some of the
proton translocation activity of the respiratory chain. As an example of of the
foregoing, we will have a look at the (in)famous ubiquinone cycle.

6.2.3 The ubiquinone cycle


Ubiquinone carries hydrogen from complexes I and II to complex III, which
in turn passes them on to complex IV. Complex III has two binding sites for
ubiquinone, and both of them are occupied while the ubiquinone cycle runs.
The cycle goes through the following steps (Figure 6.8):
1. We start with a reduced ubiquinone (QH2 ) bound to the first site, and an
oxidized ubiquinone (Q) bound to the second site.
2. The protons of QH2 are stripped off and expelled at the cytosolic side.
One electron is passed on first to a heme within complex III and then to
cytochrome C; the other electron is passed on to the second molecule of
ubiquinone.
3. The first ubiquinone, now oxidized to Q, is then replaced by a new QH2
that has been reduced in the preceding steps of the respiratory chain.
4. The protons and electrons of the new QH2 are abstracted and split as in
step 2.
5. The reduction of the second ubiquinone is now complete. It recombines
with mitochondrial protons to form QH2 .
6. Q and QH2 switch places and thereby complete the cycle.
Therefore, with each molecule of ubiquinone reduced in the respiratory chain,
the two protons it carries are expelled into the cytosol, and two additional
protons are taken up from the mitochondrial matrix and expelled into the
cytosol as well. Complex III uses ubiquinone in a dual role – as a carrier that
connects it with the preceding part of the respiratory chain, and as a prosthetic
group to facilitate the movement of protons across the membrane.
If you compare the outline of the ubiquinone cycle given here to the descrip-
tion in your textbook, you might find the similarity rather remote. In reality, as
you can see in Figure 6.4, complex III contains several more redox co-factors
that act as intermediate stepping stones in the electron transfer steps outlined
above. They have been skipped here for simplicity.
72 6 The respiratory chain

a) e-
O O
CH3O CH3 CH3O C CH3

H H
CH3O CH3O

O CH3 n O CH3 n

OH O
CH3O
2H+, e-
CH3 CH3O CH3

H H
CH3O CH3O
OH CH3 n O CH3 n

b) 2 H+
CytC

QH2 QH2 Q e-

Q Q-

2 H+
CytC

Q e- QH2

QH2 Q-

2 H+
Figure 6.8 The ubiquinone cycle, criminally simplified. a: Redox chemistry of
ubiquinone (coenzyme Q). It can accept two electrons successively and two protons
successively. b: The ubiquinone cycle (simplified so that I can understand it). Complex
III has two binding sites for ubiquinone . Oxidation of one molecule of ubiquinone
partially reduces the second one; oxidation of a new molecule of ubiquinone com-
pletes reduction of the second one and causes uptake of two protons from the cytosol.
Protons released by oxidation are at all times released at the cytosolic side.
6.2 The electron transport chain 73

6.2.4 Cytochrome C oxidase (complex IV)

Cytochrome C is a small hemoprotein that shuttles electrons from complex


III to complex IV. The latter complex, which is also known as cytochrome C
oxidase, completes the transfer of electrons by delivering them to oxygen. In
the process, it pumps some more protons out of the mitochondrial matrix. The
reduction of oxygen is the trickiest part of the entire respiratory chain, as it
takes a full 4 electrons to reduce one molecule of oxygen (O2 ) to water. Since
the electrons only arrive one at a time by way of cytochrome C, the reduction
will involve partially reduced oxygen species. These are highly toxic when let
loose upon the cell, so the enzyme must provide for stable accommodation for
all intermediate stages in the reduction.5
Cytochrome C oxidase solves this problem by clamping the oxygen between
a heme- bound iron and a histidine-coordinated copper, both of which function
as redox cofactors in the reduction of oxygen. The intermediate stages of
reduction are outlined in Figure 6.9.

6.2.5 How is electron transport coupled to proton pumping?

As pointed out above, some of the protons that are being expelled from the
mitochondrion are accepted from the hydrogen carriers NADH and ubiquinone
and travel together with electrons for a part of the journey. However, at some
point they must part company, and the protons must be expelled, whereas
the electrons are retained. Also, more protons are being expelled than can be
accounted for by the hydrogen carriers. In particular, complex IV does not
interact with any hydrogen carriers yet expels up to four protons for each pair
of electrons accepted. So, there must be mechanisms that extract energy from
the transfer of proton-less electrons and apply it towards the expulsion of
electron-less protons. How does this work?
The experimental evidence on this point is pretty fragmentary, and to the
extent they are understood the emerging mechanisms are quite complex. There-
fore, instead of trying to describe them faithfully, I will present a simplified
conceptual model to provide an idea of how things work (Figure 6.10).
The basic idea is that binding and release of electrons cause conformational
changes to a protein. This is entirely analogous to conformational changes
caused by allosteric effectors binding to enzymes, or to phosphate groups
bound to proteins (e.g. myosin light chain). An electron carries a charge, a
charge causes a field, and a field will cause forces that act on charged residues
on the protein, so it is quite easy to see how migrating electrons can cause
conformational changes.
5 The toxicity of partially reduced oxygen species such as O2– is actually exploited by phago-
cytes, which release them intra- and extracellularly to destroy microbes. Enzyme defects in the
production of these reactive oxygen species produce severe immune deficiencies.
74 6 The respiratory chain

a) R R
R R
N N
N N
R R
R R
O
O

His His
His

b)

2+ 3+ 4+ iron
e-
O O- O 2-
O O- O2- H+
H+
1+ 2+ 2+ copper
e-
H+
O2

2+ 3+ 3+ iron
2 e- 2 H+
O2- H+

2 H 2O O2- H+

1+ 2+ 2+ copper

Figure 6.9 Reduction of oxygen by cytochrome C oxidase. a: Structural features of


the active site. Iron is part of heme a3 , copper is coordinated by three histidine residues.
Oxygen is bound longitudinally between them. b: Stages of oxygen reduction. Bound
O2 is reduced to peroxide at the expense of both iron and copper. Uptake of an electron
and abstraction of a further one from iron reduces both oxygen atoms to the final level
(2– ). Uptake of protons generates first hydroxyls and then water, and uptake of further
electrons restores iron and copper to their original oxidation levels.
6.2 The electron transport chain 75

+
mitochondrial
matrix + – +

+ +
cytosol



+

Figure 6.10 A conceptual model linking electron transport to proton pumping. The
proton pump sits between two other elements of the electron transport chain. It
possesses three electron carrier cofactors (white circles). In the resting state, the
proton binding site is open to the mitochondrial matrix. Arrival of an electron at the
first carrier causes a conformational change that everts the proton binding site, so that
it now communicates with the cytosol and ejects the proton, and at the same time
facilitates migration of the electron to the next cofactor. The pump then reverts to its
resting position.

An obvious limitation of the model presented in Figure 6.10 is that it links


the transport of a single proton to the transport of a single electron. If the
number of protons is higher, we can account for it by postulating multiple
similar valves connected in series witin a single respiratory chain complex.

6.2.6 Stoichiometry of proton ejection

It is commonly stated that about 10 protons are ejected for each pair of elec-
trons abstracted from NADH, such that 4 protons are ejected at each of com-
plexes I and IV, and 2 at complex III.6 Complex II does not eject any protons
but just abstracts them from FADH2 and passes them on to ubiquinone. If you
look at Figure 6.7, you will notice that the difference in the redox potentials of
FAD and ubiquinone is rather small. Consequently, the amount of free energy
associated with the transfer of electrons from FAD to ubiquinone is too small
to permit the performance of work against the proton gradient.

6 Note that this number is at variance with the model of the coenzyme Q cycle given above.

Generally speaking, the figures given for the numbers of protons ejected at each stage vary quite
a bit between various texts.
76 6 The respiratory chain

6.3 ATP synthesis


Most of the ATP that results from complete oxidative degradation of glucose is
synthesized only after the substrate has already vanished in the form of CO2
and H2 O. At this stage, the entire available energy is stored in the so-called
proton-motive force across the inner mitochondrial membrane. ATP synthesis
is powered by the protons that yield to this force and are pulled back into the
mitochondrion.

6.3.1 The proton-motive force

The proton concentration in the cytosol is approximately ten times higher than
that in the mitochondrial matrix. How much free energy does it generate if
protons travel downhill this concentration gradient? This can be determined
from the following formula:

[H + ]in
∆G = RT ln (6.7)
[H + ]out
+
With R = 8.31 J/K mol, T = 310K, and [Hin ]/[Hout
+
] = 10 this comes to roughly
6 kJ/mol. While this is significant, the larger contribution to the proton-motive
force comes from the electrostatic membrane potential across the inner mito-
chondrial membrane. Like the proton concentration gradient, this electrical
potential is a direct consequence of the proton pumping: Each proton ejected
leaves a deficit of a positive charge, or one negative charge, inside the mito-
chondrion. In a fully energized mitochondrion, the resulting potential amounts
to 150 mV, negative inside. The free energy that this membrane potential con-
fers to each proton can again be calculated from equation 6.1 and works out to
approximately 15 kJ/mol. In summary, the proton-motive force is caused roughly
to 3/4 by the membrane potential, and to 1/4 by the proton concentration gradi-
ent.

6.3.2 ATP synthase

This complex and fascinating molecule is both an enzyme and a molecular


motor.7 It works as follows (Figure 6.11):
1. Protons flow between the a-subunit and the F0 -subunit of the ATP syn-
thase, which causes the F0 subunit and the γ-subunit attached to it to rotate
versus the rest of the molecule (Figure 6.11a).
2. The γ-subunit rubs against the inner circumference of the αβ-hexamer.
Because of the asymmetric shape of the γ-subunit, this causes the α- and
β-subunits to undergo cyclical conformational changes (Figure 6.11b).
7 A similar molecular motor drives the rotation of the flagellae found in many bacteria, which

enables them to swim. Remember that mitochondria are of bacterial origin.


6.3 ATP synthesis 77

a) Figure 6.11 Structure and func-


tion of ATP synthase. a: Side view.
δ The F0 and the wurst subunits are
rotationally mobile against the
α β
rest of the molecule. The proton
flow occurs at the interface of the
γ
a and F0 subunits. b: Top view.
The γ-subunit rubs against the α-
and β-subunits and subjects them
a F0 to cyclical changes of conforma-
tion. c: Coupling of rotation to
ATP synthesis. One of the con-
H+
formations of β accepts ADP and
phosphate, which spontaneously
b) α react to form ATP; this is driven
by the unusually high affinity of
β β
γ the next conformational state for
α α ATP. The rotation of γ forces the
β transition to the next conforma-
tion, which expels ATP and makes
β available for the next turnover.

c)

P
P
AD

ATP

ATP

3. The β-subunit, which contains the active site of the enzyme, utilizes the
energy transmitted by these conformational changes to synthesize ATP (Figure
6.11c).
How does the β-subunit transform the energy of rotation into chemical
energy for ATP synthesis? Strictly speaking, it doesn’t – it turns out that the
isolated β-subunit can create ATP all by itself. In doing so, the β-subunit adopts
distinct conformational states. In the first state, it binds ADP and phosphate.
Once both are bound, β transitions to the next state, which binds ATP with
exceptionally high affinity but no longer binds ADP and phosphate. Formation
of the several non-covalent bonds between β and ATP provides the energy that
78 6 The respiratory chain

Figure 6.12 A hypothetical model for coupling of proton flux and ATP synthase
rotation. See text for details. Source: P. Boyer, Nature 402:247–249 (1999).

is needed to form the single new phosphodiester bond in ATP.


Now with the isolated β-subunit, we have reached a dead end – ATP is
bound so avidly as to never be released, so that no further turnover can occur.
It is at this stage that the γ-subunit comes into play. Rotation of γ forces
another change of conformation upon β that in turn kicks out the ATP of the
active site (Figure 6.11c). The force applied by γ on β must be so strong as to
overcome and offset the large binding energy that ties the ATP to the enzyme.
In summary, the formation of ATP proceeds spontaneously inside β, and the
energy of rotation is applied to force out the avidly bound ATP and reset β for
the next round of catalysis.
The last remaining puzzle is how the proton flux actually promotes rotation
of the F0 - and γ-subunits. A hypothetical model is illustrated in Figure 6.12. The
F0 -subunit consists of 10 c-chains arranged in a pie-slice fashion. Each of them
has a strategic aspartate residue that faces the surrounding lipid membrane.
When a given c-chain encounters the a-subunit, the aspartate first connects
to a proton-conduction channel (across a) that allows it to discharge a proton,
which it had picked up during the last rotation, and then to another channel
that causes it to accept a new proton from the cytosol.
While there is indeed evidence for alternating accessibility of several strate-
gic amino acid residues from the two opposite sides of the membrane, this
model has a problem: While it tells us how rotation of ATP synthase could
dissipate the proton gradient, it does not tell us how ATP synthase actually
derives any torque from this, so that it can perform work against resistance.
More specifically, why would the rotor always keep going in the same direction
6.4 The ATP yield of oxidative glucose degradation 79

instead of just oscillating? This would save it the trouble of working while
still permitting proton flux. A steam engine gets around a similar problem by
inertia, which is created by adding a nice, heavy, cast-iron flywheel. That’s not
possible here, because of the minuscule dimensions.8 Accordingly, there must
be a tighter coupling of proton flux and rotation that most likely involves some
conformational flexibility in both the a and the c subunits. There are some
experimental data to support this assumption.
There is, however, one interesting finding that the model in Figure 6.12 does
account for: The number of protons transported per rotation is identical to
that of the c subunits in F0 . Intriguingly, ATP synthases in different organisms
vary in their number of c subunits. This will directly affect the stoichiometry of
ATP synthesis and proton transport. It would be interesting to know whether
there are complementary variations in the number of protons driven out per
electron during electron transport. If not, the different subunit stoichiometry
of F0 should directly translate into a different ATP yield in the entire respiratory
chain.9

6.4 The ATP yield of oxidative glucose degradation

We can now determine how much ATP is produced through the complete oxi-
dation of glucose via glycolysis, TCA, and respiratory chain. Here are the bits
and pieces:
1. For each molecule of glucose, a total of 10 moles of NADH and 2 FADH2
are produced by glycolysis, pyruvate dehydrogenase, and citric acid cycle.
2. 10 protons are exported per NADH, and 6 per FADH2 in the respiratory
chain, for a total of 112 protons.
3. Each revolution of ATP synthase consumes 10 protons and generates 3
ATP.
Overall, we obtain 112 protons per glucose × 3 ATP per 10 protons, or
33.6 ATP per molecule of glucose. Therefore, 33.6 ATP could be generated
in the respiratory chain for each molecule of glucose degraded, if all protons
were available for driving ATP synthase. However, some protons are diverted
to other purposes, so that the actual yield will be lower than this theoretical
value. Most importantly, some protons are needed for ATP transport. ATP
synthesized in the mitochondrion needs to be exported to the cytosol, and
ADP produced there needs to get back in. This is accomplished by a special

8 A student who took this class in 2005, Kelvin Cheung, took up the challenge to calculate the

kinetic energy of rotating ATP synthase; it works out to about one billionth of the energy required
for making 1 ATP. Honorable mention.
9 If you are interested in this problem, you are welcome to dig up and some recent information

on this and discuss it with me. If it merits inclusion here, it will earn you an honorable mention
and a recommendation letter.
80 6 The respiratory chain

transporter protein in the inner mitochondrial membrane that exchanges ATP


and ADP for each other.
Since ATP carries one more negative charge than ADP (ATP4– vs. ADP3– ), this
exchange amounts to a net export of one negative charge, or to the net import
of one positive charge per ATP. The total need of protons per ATP synthesis
plus export to the cytosol is therefore approximately 4,10 so that the actual
ATP/glucose ratio is closer to 28 than to 33. Together with the 4 molecules of
ATP and GTP generated in glycolysis and the TCA, the overall yield of ATP per
molecule of glucose is approximately 32.
It is worth noting that the extra proton expended on the export of ATP is
not ‘wasted’, as some textbooks lament, but can be considered well spent. It
enables the transport of ATP and ADP against their concentration gradients,
allowing for the maintenance of a high ATP/ADP ratio in the cytosol, which will
help all ATP-consuming reactions there to go at speed, and a higher ADP/ATP
ratio in the mitochondrion, which will help the ATP synthase to go at speed. It
is kind of like driving on the highway: Sure, you could save fuel by driving at
60 km/h all the time, but would you?

6.5 Auxiliary shuttle systems for the re-oxidation of cy-


tosolic NADH
In chapter 3, it was mentioned that under aerobic conditions the NAD+ con-
verted to NADH by glyceraldehyde-3-phosphate dehydrogenase is re-oxidized
in the respiratory chain. However, NADH cannot pass the inner mitochondrial
membrane, and in fact not even the more porous outer membrane. So, how
then is its oxidation accomplished?
It turns out that NADH is not translocated at all but is re-oxidized, or dehy-
drogenated, in the cytosol. The hydrogen is then brought to the mitochondrion
by other carriers. This is accomplished by several shuttle systems, in a some-
what roundabout manner. The shuttles tie together several enzyme activities
with specific transporters in the inner mitochondrial membrane (Figure 6.13).
One enzyme that regenerates cytosolic NAD+ is cytosolic malate dehydroge-
nase, which reduces oxaloacetate to malate. Cytosolic malate is then exchanged
for mitochondrial α-ketoglutarate by a specific transporter and dehydrogenated
back to oxaloacetate inside the mitochondrion.
The question whether or not oxaloacetate can leave the mitochondrion in
exchange for α-ketoglutarate is, in my opinion, not settled. If it can, it is
possible to draw a pretty simple shuttle mechanism, shown in Figure 6.13a. If
it cannot, we have to use transamination (see section 12.2) as a workaround:
10 It is not actually a proton; therefore, in my understanding, it is only the membane potential

component but not the proton concentration component of the proton- motive force that gets
dissipated. Then, it costs about 3/4 of the energy of a pumped proton to export one ATP.
6.5 Shuttle systems for the NADH re-oxidation 81

a) Figure 6.13 Shuttle


Malate Malate systems for the transfer
of NADH equivalents
NAD+
NAD+ from the cytosol to the
α-Ketoglutarate α-Ketoglutarate mitochondrion. a: A
NADH+H+ fairly simple, hypothetical
NADH+H+
shuttle that is not in the
Oxaloacetate Oxaloacetate
textbooks. b: The malate-
aspartate shuttle. c: The
glycerophoshate shuttle.
b) NAD+ NAD+ Abbreviations: DHAP, dihy-
droxyacetone phosphate;
NADH+H+ Malate Malate NADH+H+ GPD, glycerolphosphate
dehydrogenase. Continu-
Oxaloacetate Oxaloacetate
ous gray bars represent
α-Ketoglutarate α-Ketoglutarate the inner mitochondrial
membrane; the broken bar
Glutamate Glutamate in c represents the outer
mitochondrial membrane.
See text for further details.
Aspartate Aspartate

c)

IV

C
III
Glycerol-P Glycerol-P
Q
NAD+ 2 e-
GPD
(FAD)
NADH+H+
II
DHAP DHAP

I
2 H+

Oxaloacetate is transaminated using mitochondrial glutamate and the resulting


aspartate exchanged for cytosolic glutamate. In the cytosol, transamination is
reversed, which closes the cycle. This textbook-approved cycle is known as the
malate-aspartate shuttle (Figure 6.13b).
In the glycerolphosphate shuttle, the hydrogen is never actually transported
to the mitochondrion. Dihydroxyacetonephosphate serves as the intermedi-
ate hydrogen acceptor and is reduced in the cytosol to glycerolphosphate by
glycerolphosphate dehydrogenase. Glycerolphosphate traverses the outer mi-
tochondrial membrane and reaches the surface of the inner one, where it is
82 6 The respiratory chain

converted back to dihydroxyacetonephosphate by a second dehydrogenase,


which abstracts the electrons and feeds them into the respiratory chain at the
level of ubiquinone (Figure 6.13c). This is similar to the activity of succinate
dehydrogenase, and as with the latter, FAD is the coenzyme employed by the
mitochondrial glycerolphosphate dehydrogenase.
The glycerolphosphate shuttle bypasses complex I in the respiratory chain
and therefore induces ejection of four fewer protons from the cytosol. How-
ever, this shortfall is partially compensated for by the two protons that stay
behind in the cytosol (or more accurately, the intermembrane space) when the
electrons get abstracted from glycerolphosphate. While this shuttle is some-
what less energy-efficient than the malate-aspartate shuttle, it certainly is more
straightforward than the latter, since it avoids all substrate transport across the
inner mitochondrial membrane. Remember that inside the mitochondrion the
free concentration of oxaloacetate is low; thus, oxaloacetate probably forms
the bottleneck in the malate-aspartate shuttle. It is interesting to note that
the glycerolphosphate shuttle is highly active in insect muscle, which has an
extremely high ATP turnover during flight.

6.6 Regulation of the respiratory chain


Most of the time and in most cells, the respiratory chain runs at rates that are
substantially below the maximal rate. How is the flow through the respiratory
chain controlled? In a healthy and not maximally exerted cell, there is much
more ATP than ADP or phosphate, so that these become limiting for the flow.
If ATP synthase is short of substrates, the proton-motive force will not be
dissipated, so that the proton pumps will have a harder time to extrude more
protons and will eventually stall. Since electron transport and proton pumping
are tied to one another, this means that dehydrogenation of NADH and FADH2
will stall as well.
The flow rate of the respiratory chain is also coupled to those of the pre-
ceding pathways of glycolysis and the TCA. Such coupling occurs by negative
feedback at various levels:
1. A low flux through the respiratory chain will lead to the accumulation
of NADH, which slows down glyceraldehyde-3-phosphate dehydrogenase, pyru-
vate dehydrogenase, and the NAD+ -dependent isocitrate dehydrogenase.
2. A low consumption of ATP will result in its accumulation to higher levels.
Many enzymes, including phosphofructokinase, are inhibited by ATP.
These regulatory mechanisms are reasonably straightforward. There is,
however, one remaining mystery. We have already noted that there are two
forms of isocitrate dehydrogenase, one using NAD+ and the other NADP+ as
the cosubstrate. While the NAD+ -dependent form is inhibited by NADH and
ATP, the NADP+ -dependent form, which is actually the more highly expressed
one of the two, is not subject to such inhibition. This would suggest that it
6.6 Regulation of the respiratory chain 83

might go at full blast even when the demand for ATP is low and NADH is
high! How, then, is this enzyme prevented from uncontrolled consumption of
isocitrate?
It appears that, at least during times of low demand for ATP, NADP+ -
dependent isocitrate dehydrogenase is close to equilibrium. This equilibrium is
sustained by a high mitochondrial level of NADPH, which in turn is maintained
by NAD+ /NADPH transhydrogenase. This remarkable protein, wich is located
in the inner mitochondrial membrane, is both an enzyme and a transporter. It
reduces NADP+ to NADPH at the expense of NADH. As with ATP synthase, the
enzyme reaction is coupled to the translocation of protons:

NADH + NADP+ + H+ + +
out → NAD + NADPH + Hin (6.8)

Figure 6.14 shows how the function of transhydrogenase is integrated with


the function and regulation of the TCA and the respiratory chain.11
When the demand for ATP is low, NADH and the proton-motive force will
both be at high levels, which will cause the transhydrogenase to reduce NADP+
at the expense of NADH. At equilibrium, the NADPH concentration will be
high, which results in near-equilibrium conditions for the NADP+ -dependent
isocitrate dehydrogenase also. There may, however, be a low net flux within an
interesting futile cycle, which involves the two isocitrate dehydrogenases and
the transhydrogenase (Figure 6.14a). The net effect of this cycle is the influx of
one proton in each round.
On the other hand, when the demand for ATP is high, the proton-motive
force and the level of NADH will be lower. Under these conditions, the tran-
shydrogenase will switch direction, now consuming NADPH to produce more
NADH, which will be consumed at high rate in the respiratory chain. At the
same time, transhydrogenase will work as an auxiliary proton pump, thus di-
rectly contributing to the proton-motive force. This will reduce the level of
NADPH, which in turn will topple the equilibrium of NADP+ -dependent isoci-
trate dehydrogenase in favour of supplying more NADPH, which will help to
keep the entire process going.
Now that is a marvelous piece of engineering by Mother Nature, isn’t? This
is as close as it gets to intelligent design.

11 This is my take on the subject. There is, however, considerable variety of opinion on the role

of this fascinating enzyme.


84 6 The respiratory chain

a) Isocitrate Ketoglutarate + CO2

mitochondrial
matrix
NADPH NAD+
H+

NADP+ NADH respiratory chain

H+ cytosol

b) Isocitrate Ketoglutarate + CO2

NADPH NAD+
H+

NADP+ NADH respiratory chain

H+
Figure 6.14 Function of NADP+ /NADH transhydrogenase, and its integration with the
TCA and the respiratory chain. a: Function of transhydrogenase in idling mode, while
the demand for ATP is low. In this situation, the extra-mitochondrial H+ concentration
is high. Protons enter through transhydrogenase and sustain a high concentration
of NADPH, some of which in turn is dissipated by NADP+ -dependent isocitrate dehy-
drogenase. b: Function of transhydrogenase at times of high demand for ATP. A high
throughput of the respiratory chain causes the extra-mitochondrial H+ concentration to
drop. The proton flow through transhydrogenase now reverses, as does the operation
of NADP+ -dependent isocitrate dehydrogenase. This results in the provision of extra
NADH for the respiratory chain, as well as protons for ATP synthesis.
Chapter 7

Gluconeogenesis

The metabolic pathways we have considered so far account for the complete
oxidative degradation of glucose. Glucose is the most important substrate of
energy metabolism for several reasons:
1. Glucose accounts for a large share of all calories in a typical human diet.
2. Some cell types, for example erythrocytes, depend entirely on it for all
their metabolic energy. Others, such as nerve cells, only very reluctantly give
up their preference for it, althougy they can adapt to ketone bodies (which
are formed by fatty acid degradation) as alternative substrates in times of
starvation.
3. Glucose is the major source of NADPH, which is needed for many biosyn-
thetic tasks, including fatty acid and sterol synthesis. The pathway that regen-
erates NADPH by oxidation of glucose—the hexose monophosphate shunt—will
be discussed later on.
It is plausible then that a pathway should exist that can turn other sub-
strates into glucose if the latter is lacking in the diet. In fact, many carnivorous
mammals and many humans—and not merely decadent Westerners,think of the
traditional lifestyle of Inuit—live on diets that are very rich in protein but not
glucose. Such diets will typically also be rich in fat; however, fatty acids cannot
be converted to glucose in the mammalian metabolism. Amino acids therefore
are the major source of carbon for the synthesis of glucose. The pathway of
glucose synthesis is called gluconeogenesis.

85
86 7 Gluconeogenesis

Figure 7.1 Overview of gluconeo- Glucose


genesis and its role in human 4
metabolism. Lactate and amino Glucose-6-P
acids that can be converted to
pyruvate or to TCA intermediates Fructose-6-P
serve as carbon sources. The con- 3
version to glucose uses the re- Fructose-1,6-bis-P
versible reactions from glycolysis,
and 4 distinct reactions that cir- Dihydroxyacetone-P +
cumvent the ones from glycolysis Glyceraldehyde-P
that are irreversible. These re-
actions are catalyzed by pyruvate 1,3-Bis-P-glycerate
carboxylase (1), phosphoenolpyru-
vate carboxykinase (2), fructose-1,6-
3-P-glycerate
bisphosphatase (3) and glucose-6-
phosphatase (4).
P-enolpyruvate
2
Pyruvate Lactate
1
CO2
Acetyl-CoA

Oxaloacetate Citrate

Malate Isocitrate
CO2
Fumarate α-Ketoglutarate
CO2
Succinate Succinyl-CoA

Amino acids

Gluconeogenesis is essentially confined to two organs: The liver and the


kidney. Both organs have an ample supply of amino acids: The liver from the
intestine via the portal vein, the kidneys because they extract amino acids from
the considerable volume (~150 l/day) of plasma ultrafiltrate that represents the
first stage of urine secretion (see section 14.6).

7.1 Reactions in gluconeogenesis

Gluconeogenesis converts pyruvate and oxaloacetate to glucose. It is essentially


a reversal of glycolysis, with workarounds for those reactions of glycolysis that
are energetically irreversible. These four reactions are highlighted in Figure 7.1.
7.1 Reactions in gluconeogenesis 87

a) Figure 7.2 The pyruvate carboxylase re-


O action. a: The enzyme contains a biotin
coenzyme covalently attached to a lysine
HN NH
residue. The long, flexible arm allows the
coenzyme to interact with multiple active
H
N sites. b: In the first step of the reaction,
S Enzyme biotin is carboxylated. The carboxyl group
O is obtained from a bicarbonate ion (top
left) and requires ATP. Carboxyphosphate
(top right) is likely formed intermittently.
c: In the second step (which occurs at a
b) ADP
O separate active site), the carboxyl group is
ATP
O O P O transferred onto pyruvate. This step re-
O O O quires abstraction of a proton from the
OH OH pyruvate methyl group to yield a carban-
ion.
Pi
O O
O
HN N C HN NH
OH

R R
S S

c) O
O
Enzyme-B H3C C C
O

O
O
Enzyme-BH+ H2C C C
O
Biotin-CO2

Biotin
O O O
C C C
H2
O O

The final reaction in glycolysis is the transfer of the phosphate group from
phosphoenolpyruvate (PEP) to ATP. This reaction is irreversible because of the
strongly exergonic nature of the accompanying rearrangement of pyruvate from
the enol to the keto form (see section 3.4.4). In gluconeogenesis, it takes two
enzymatic steps to turn pyruvate back into PEP: (1) Carboxylation to oxaloac-
etate by pyruvate carboxylase (Figure 7.2), and (2) conversion of the latter to
PEP by phosphoenolpyruvate carboxykinase (Figure 7.3a).
Pyruvate carboxylase requires biotin as a coenzyme. This coenzyme is
88 7 Gluconeogenesis

flexibly attached to the enzyme (Figure 7.2a) in a manner reminiscent of lipoic


acid in pyruvate dehydrogenase (see Figure 5.2), and for a similar reason: The
reaction occurs in separate steps at different active sites, which with some
biotin-dependent enzymes (though not with pyruvate carboxylase) are located
on separate enzyme subunits.
Biotin serves as an intermediate carrier of a carboxyl group that is generated
from bicarbonate, which is equivalent to CO2 . Remarkably, therefore, we are
able to metabolically fix CO2 , just like plants! Before you try to claim Kyoto
treaty credits for this ability, however, it is necessary to consider that the very
same molecule of CO2 gets released again in the next step. The whole purpose
of its transient fixation consists in the facilitation of the subsequent reaction,
which is outlined in Figure 7.3a.

7.2 Glucogenic amino acids and gluconeogenesis


So far, we have seen that the TCA serves to completely degrade acetyl-CoA. In
addition to this degradative function, the TCA also serves to collect the carbon
skeletons of several amino acids, which can be converted into different TCA
intermediates (Figure 7.1) and from there to oxaloacetate. Other amino acids
can be converted to pyruvate. Since pyruvate can be converted to oxaloacetate,
we can summarize that all amino acids that can be converted to any interme-
diate of the citric acid cycle or to pyruvate can be utilized for gluconeogenesis.
Such amino acids are referred to as glucogenic. Amino acids that cannot be
converted to glucose but can be converted to ketone bodies instead are referred
to as ketogenic. We will learn more about both amino acid degradation and
ketone body formation later.

7.3 Regulation of gluconeogenesis


7.3.1 The need to control futile cycling
Figure 7.3b compares the reactions of the two phosphatases involved in glu-
coneogenesis with the two corresponding kinases that operate in glycolysis.
What would happen if, for example, hexokinase and glucose-6-phosphatase
were active at the same time? Let’s write it down:

Glucose + ATP ------→


- Glucose-6-phosphate + ATP
Glucose-6-phosphate+H2 O ------→
- Glucose + phosphate

We can combine these two equations and cancel glucose and glucose-6-
phosphate, since they occur on both sides. We obtain:

ATP + H2 O ------→
- ADP + phosphate (7.1)
7.3 Regulation of gluconeogenesis 89

a) O O
CO2
O
C C H2C C
H2
O OH OH
O O
O

N
NH
O O O HC
O
H2
H2C C O P O P O P O C N
O N NH2
OH
O O O O HC CH

O P O HC CH

OH OH OH

b) Fructose-1,6-bis-phosphate
H 2O
1 phosphate
Fructose-6-phosphate
2

Glucose-6-phosphate
H 2O
3
phosphate
Glucose

Figure 7.3 Further reactions in gluconeogenesis. a: PEP carboxykinase uses GTP to


convert oxaloacetate to PEP. The carboxylate group serves to facilitate the transient
formation of the enolate anion. b: Fructose-1,6-bisphosphatase (1) and glucose- 6-
phosphatase (3) revert the two initial phosphorylations that occur in glycolysis. They
do no regenerate ATP but simply remove the phosphate groups by hydrolysis, which is
an exergonic reaction. 2: Phosphohexoseisomerase.

This means that the simultaneous activity of the two enzymes would cause
unrestricted, idle hydrolysis of ATP. A pathway with no other effect than the
expenditure of ATP (or some other form of metabolic energy) is called a futile
cycle. Futile cycling will also result from simultaneous activity of of fructose-
1,6-bisphosphatase with phosphofructokinase, and of pyruvate kinase with
pyruvate carboxylase and PEP carboxykinase, with the latter differing only by
hydrolysing GTP instead of ATP.
It is clear that unchecked operation of futile cycles would be a disaster – all
ATP would be rapidly consumed, and the energy simply be dissipated as heat.
Of note, the liver has a temperature well above the body temperature (39-40 ◦ C),
and a fair share of the process heat generated in liver metabolism is believed
90 7 Gluconeogenesis

to derive from the futile cycles just discussed. Still, there are regulatory mecha-
nisms to keep these cycles in check. While this applies to all enzymes involved,
as an example we will consider those that concern phosphofructokinase and
fructose-1,6- bisphosphatase.

7.3.2 ATP, ADP and AMP in the regulation of phosphofructokinase and


fructose-1,6- bisphosphatase
Phosphofructokinase is inhibited by ATP. This makes sense, since ATP forma-
tion is the main purpose of glycolysis (in conjunction with the TCA and the
respiratory chain). On the other hand, depletion of ATP will result in a buildup
of ADP and AMP; it therefore also makes sense that ADP and AMP stimulate
phosphofructokinase (section 2.5). While most ATP-consuming reactions yield
ADP rather than AMP, AMP is formed from ADP by adenylate kinase:

2 ADP ------→
- ATP + AMP

This allows for a makeshift regeneration of some ATP from ADP. Also, if we
consider the equilibrium:

[ATP] [AMP] K [ADP]2


K= 2 a [AMP] =
[ADP] [ATP]

we see that [AMP] will vary with the square of [ADP]; [AMP] therefore is the
more sensitive parameter to detect changes in the availability of ATP.
Fructose-1,6-bisphosphatase is stimulated by ATP and inhibited by AMP.
This behaviour is opposite to that of phosphofructokinase, and it ensures that
one enzyme will be active when and only when the other one is inactive, so that
futile cycling and ATP hydrolysis is avoided.

7.3.3 Hormonal control of phosphofructokinase and


fructose-1,6-bisphosphatase
Both enzymes also show opposite responses to another molecule, fructose-2,6-
bisphosphate (Figure 7.4b). This molecule is not a regular metabolite of glucose
metabolism but is synthesized solely for the sake of regulation, at levels much
lower than fructose-1,6- bisphosphate. Its concentration is under the control
of hormones via the secondary messenger 3’,5’-cyclo-AMP (cAMP). The entire
cascade consists of the following stages (Figure 7.4c):
1. The hormones bind to their respective receptors on the cell surface,
which then promote formation (glucagon and epinephrin) or degradation (in-
sulin) of cAMP.
2. cAMP binds to and activates protein kinase A, which phosphorylates the
enzyme phosphofructokinase 2. Phosphorylation can be reversed by a protein
phosphatase.
7.3 Regulation of gluconeogenesis 91

a) Pi Fructose-6-P ATP

+ ATP -
Fructose-1,6 Phospho-
bisphosphatase - AMP + fructokinase

- Fructose-2,6-bis-P +

H 2O Fructose-1,6-bis-P ADP

b) O O O
O P OH O P OH O P OH
O O O OH
CH2 O H2C CH2 O H2C
OH OH
OH O
OH OH HO P O
O

c) Epinephrin,glucagon Insulin
+
+

ATP cAMP AMP


+

Protein kinase A
ATP ADP
P

PFK-2/(bis-P’ase) (PFK-2)/bis-P’ase

Fructose-2,6-bis-P

Fructose-6-P

Figure 7.4 Regulation of phosphofructokinase and of fructose-1,6-bisphosphatase.


a: Allosteric effectors have opposite effects on the two enzymes. b: Structure of
fructose-2,6-bisphosphate (right). Fructose-1,6-bisphosphate is shown for comparison.
c: Control of fructose-2,6-bisphosphate levels by hormones. See text for details.
92 7 Gluconeogenesis

3. Phosphofructokinase 2 has two opposite activities: In the dephospho-


rylated state, it acts as a kinase and therefore increases the level of fructose-
2,6-bisphosphate. In the phosphorylated state, it acts as the corresponding
phosphatase and therefore lowers the level of fructose-2,6- bisphosphate.
So, what is the upshot of these hormone actions? Let’s go over it step by
step again. Here is the effect of glucagon and epinephrin:

Glucagon ↑ or epinephrin ↑ → cAMP ↑ →


PFK-2 ↓, fructose-2,6-bisphosphatase ↑ → fructose-2,6-bisphosphate ↓ →
gluconeogenesis favoured

This is the effect of insulin:

Insulin ↑ → cAMP ↓ →
PFK-2 ↑, fructose-2,6-bisphosphatase ↓ → fructose-2,6-bisphosphate ↑ →
glycolysis favoured

Thus, glucagon and epinephrine promote gluconeogenesis, inhibit glycolysis


and so increase availability of glucose, while insulin has the opposite effects and
promotes the net consumption of glucose. It should be noted that ATP and AMP
adjust the activity of phosphofructokinase and fructose-1,6-bisphosphatase ac-
cording to the intracellular situation, whereas the hormones, via cAMP and
fructose-2,6-bisphosphate, control the same enzymes on behalf of the metabo-
lism of the body as a whole. This is a nice example of how diverse regulatory
signals are integrated at the molecular level.

7.4 Energy balance of gluconeogenesis


In the formation of pyruvate from glucose—that is, glycolysis—there was a net
gain of two molecules of ATP per molecule of glucose. How many molecules of
ATP are required to revert the process?
• Going from pyruvate to phosphoenolpyruvate via oxaloacetate costs one
ATP and one GTP, which is equivalent to 2 ATP.
• A third ATP is spent in the (reversible) phosphorylation of 3- phospho-
glycerate to 1,3-bisphosphoglycerate by phosphoglycerate kinase.
• We need two molecules of pyruvate for one molecule of glucose; this
works out to six molecules of ATP per molecule of glucose.
If glycolysis were reversible without change (which it is not), then only two
molecules of ATP would have to be consumed. The expenditure of 4 extra
molecules of ATP is necessary to revert the energy balance of the pathway so
that it actually proceeds into the opposite direction. Formation of no more than
two ATP molecules makes it exergonic to turn glucose into pyruvate, whereas
expenditure of extra ATP makes it exergonic to turn pyruvate back into glucose.
7.5 The Cori cycle 93

glucose glucose

2 ADP NAD+ 6 ADP


NAD+
2 ATP NADH+H+ 6 ATP
NADH+H+
pyruvate lactate pyruvate

Figure 7.5 The Cori cycle. Lactate generated from glucose by anaerobic glycolysis in
the skeletal muscle during exercise is moved to the liver, reoxidized to pyruvate and
turned back into glucose by gluconeogenesis, which is then returned to the muscle or
other peripheral tissues.

7.5 The Cori cycle


We have seen in section 3.6 that lactate accumulates in anaerobic glycolysis.
Cells producing lactate will release it into the blood. What becomes of it subse-
quently? It may be taken up by the liver, reoxidized to pyruvate, and fed back
into gluconeogenesis. The combination of glycolysis in peripheral tissues with
gluconeogenesis in the liver is referred to as the Cori cycle (Figure 7.5).
While gluconeogenesis is certainly important in the utilization of lactate
generated in the skeletal muscle, most books fail to point out that the two
stages of this ‘cycle’ cannot be active at the same time, for the following reasons:
1. Under conditions of maximal exercise, liver perfusion is minimized, and
the liver itself is quite as anaerobic as the muscle itself. It gets even less oxygen
than the muscle does, and without oxygen, the liver cannot regenerate the NAD+
needed for turning lactate into pyruvate any more than the muscle could.
2. Since gluconeogenesis costs more ATP than glycolysis generates (section
7.4), the net energy balance of the Cori cycle will be the expenditure, not the
gain of ATP – not helpful for sustaining maximal exercise.
So, what really happens is that after maximal exercise has stopped, the liver
slowly scoops up the lactate that has accumulated in the blood and turns it
back into glucose. Thus, the Cori cycle operates asynchronously rather than
continuously – it has more similarity with the hog cycle of the agricultural mar-
kets than with a regular metabolic cycle such as the TCA. The Cori cycle does
94 7 Gluconeogenesis

indeed run synchronously with cells such as erythrocytes and thrombocytes,


which don’t have mitochondria and thus rely completely on anaerobic glycol-
ysis even under aerobic conditions. However, their energy requirements are
minuscule compared to those of skeletal muscle.

7.6 The glyoxylate cycle


It was stated above that fatty acids cannot be utilized for gluconeogenesis. Also,
it was mentioned earlier that fatty acids are degraded via acetyl-CoA. Since
acetyl-CoA is degraded via the TCA, and TCA intermediates can be utilized for
gluconeogenesis, why is it not possible to turn fatty acids into glucose? This is
because of the different roles of the TCA intermediates in the two processes:
In the degradation of acetyl-CoA, they have a catalytic role – they are re-
quired for the cycle to run, but there is no net gain or loss of intermediates.
Acetyl-CoA fed into the TCA is consumed without increasing the pool of TCA
intermediates. Now, in gluconeogenesis, oxaloacetate is drained from the pool
of TCA intermediates. Thus, the TCA can only sustain gluconeogenesis to the
extent it is supplied with new intermediates from other sources.
To turn fatty acids into glucose, we therefore need a means to use acetyl-
CoA for increasing the pool of intermediates. Such a pathway does not exist in
mammals, but it does exist in plants; it is known as the glyoxylate cycle. It is a
side-road to the TCA that allows them to use two molecules of acetyl-CoA per
cycle for the net synthesis of one C4- intermediate. Two reactions are required
for this cycle (Figure 7.6):
1. Isocitrate is split into succinate and glyoxylate by isocitrate lyase. Since
the isocitrate dehydrogenase and the α-ketoglutarate dehydrogenase reactions
are bypassed, the loss of two carbons as CO2 is avoided; these carbons are
retained in the form of glyoxylate.
2. Glyoxylate combines with the second acetyl-CoA to form one molecule of
malate. This reaction is catalysed by malate synthase, and like the citrate syn-
thase reaction it is pushed forward by the concomitant hydrolysis of coenzyme
A.
You know that many plant seeds are very rich in oil (=fat). The glyoxylate
cycle enables plant seeds to store metabolic energy and carbon as fat, and to
use it for the synthesis of glucose and other carbohydrates during germination.
Fat is water-free and has approximately twice the content of metabolic energy
per gram of carbohydrates; it is therefore both more compact and also more
resistant to microbial degradation than starch.

7.7 Additional roles of gluconeogenetic enzymes


Two enzymes that occur in gluconeogenesis have important roles in other
metabolic pathways. One of them is glucose-6-phosphatase. Glucose-6-phosphate
7.7 Additional roles of gluconeogenetic enzymes 95

a) H2C COOH H2C COOH


Isocitrate lyase
HC COOH C COOH
H2
HO C COOH
H O C COOH
H

Glyoxylate

O O

H3C C S CoA H2 C C OH HS CoA


Malate synthase
+
O C COOH HO C COOH
H H

b) Glucose
Acetyl-CoA

Citrate
Oxaloacetate
Isocitrate
Malate
1 Glyoxylate
Fumarate

Acetyl-CoA
Succinate 2

Figure 7.6 The glyoxylate cycle of plants. a: Two enzyme reactions are required in
addition to those of the TCA. Both reactions are similar to the citrate synthase reaction.
b: Overview of the cycle. Dotted arrows represent reactions of the TCA that are skipped
by the glyoxylate cycle. (The conversion of oxaloacetate to glucose has been abridged.)

can be generated not only by gluconeogenesis but also during degradation of


glycogen, the polymeric storage form of glucose (see chapter 8). Glucose-6-
phosphate released from glycogen likewise must undergo dephosphorylation
to glucose by glucose-6-phosphatase before release into the bloodstream.
The other enzyme that deserves mention is pyruvate carboxylase. If you
look at Figure 1.2, you will see that pyruvate can enter the TCA via two routes:
As a substrate, after conversion to acetyl-CoA, and as a C4-intermediate, namely
oxaloacetate. If a shortage of TCA intermediates occurs, acetyl-CoA will back
up. Acetyl-CoA allosterically activates pyruvate carboxylase, which produces
oxaloacetate and helps restore the level of TCA intermediates back to normal.
Pyruvate carboxylase can also help to provide NADPH, which otherwise is
provided prominently by the hexose monophosphate shunt. Oxaloacetate is
96 7 Gluconeogenesis

reduced to malate by malate dehydrogenase—as we have seen, this reaction


occurs both in mitochondria and in the cytosol—and malate is then cleaved by
malic enzyme, which yields pyruvate and NADPH. This reaction is considered
in more detail in section 12.1.
Chapter 8

Glycogen metabolism

Glycogen is a polymeric storage form of glucose (Figure 8.1). It is very similar


to amylopectin, the branched polyglucose molecule found in starch (Figure 1.6);
the only difference is that glycogen is more highly branched. It is synthesized
from glucose in times of plenty, that is after a meal rich in carbohydrates, and
converted back to glucose when the later is in demand. It is found in many
tissues, but only two of these are quantitatively important:
1. The liver stores glycogen for the purpose of releasing the glucose con-
tained in it into the circulation; glycogen stored here therefore serves the entire
organism. The glycogen content of a fully stocked liver amounts to as much as
10% of its wet weight, that is about 150-200 grams. Since glycogen is so similar
in structure to starch, this is comparable to 200 grams of dry spaghetti.
2. According to the books, the skeletal muscle stores glycogen only for its
own use – glucose released from glycogen in the muscle is not released into the
blood but consumed then and there. The concentration of glycogen in muscle
tissue is lower than in the liver, but because of the large mass of sceletal muscle
the amount of glycogen stored in muscle is actually about twice that found in
the liver.
The alleged inability of skeletal muscle to release glucose would result from
a lack of the enzyme glucose-6-phosphatase, without which free glucose cannot
be obtained from glycogen. However, recent work has shown that this enzyme
is indeed expressed at appreciable levels in the muscle also.1 This raises the
1 See Shee et al., J Biol Chem 279:26215-9 (2004) and references cited therein.
97
98 8 Glycogen metabolism

O O O O O

CH2

O O O O O O

O O O O O

CH2 CH2

O O O O O O O O-Tyr Glycogenin

Figure 8.1 Structure of glycogen: A single α-1→4-polymer of glucose is attached to


the protein glycogenin. Additional polymers branch of from the first one and from
other branches. Two adjacent branching points are not 2 (as shown here) but 6-8
glucose residues apart. Attachment of the branches is by α-1→6-glycosidic bonds. The
total number of glucose residues in the entire polymer can be as high as 105 .

interesting possibility that, contrary to traditional belief, skeletal muscle does


function as a reservoir of glucose. This is important, since an accurate un-
derstanding of blood glucose regulation and homeostasis is essential in the
treatment of diabetes mellitus.
Why do organisms store glucose in polymeric rather than in free form? Free
glucose would cause an inacceptably high osmotic pressure inside the cell. The
osmotic pressure associated with a solute follows the gas equation:
n
pV = nRT a p = RT
V
This means that the osmotic pressure is proportional to the number of
molecules per volume, or the molar concentration. Consider the amount of
glycogen stored in the liver: 10% equals 100 g/l. Each glucose residue in glycogen
has a molar weight of 162 Da, which works out to roughly 0.6 mol/l glucose
residues. This is approximately twice as high as the total concentration of small
solutes inside the liver cell. Therefore, if all the glycogen were converted to
glucose, the osmotic pressure would triple, and the liver cell would suck water
like a delirious camel and burst. Polymerization of glucose vastly reduces its
molar concentration and therefore its osmotic activity; it makes storage of large
amounts of glucose ‘bio-compatible’.

8.1 Glycogen synthesis


The following enzyme reactions occur in glycogen synthesis (Figure 8.2):
1. Glucose activation,
2. Initiation of glycogen synthesis,
8.1 Glycogen synthesis 99

3. Chain elongation,
4. Introduction of branch points.

8.1.1 Glucose activation


Glucose activation consists in the formation of UDP-glucose from glucose-6-
phosphate, which to this end is converted to glucose-1-phosphate by the en-
zyme phosphoglucomutase. Glucose-1-phosphate is then activated to UDP-
glucose by glucose-1-phosphate uridylyltransferase; this reaction uses uridine
triphosphate (UTP) and releases pyrophosphate.2

8.1.2 Initiation and elongation


The first molecule of glucose is attached to the OH group of a tyrosine residue
in the small protein glycogenin, which therefore serves as a seed for glycogen
synthesis. Subsequently, another glucose subunit is attached to the 4-OH group
of the first one, and this process is then repeated over and over, giving rise to a
long, linear polysaccharide. UDP-glucose is the substrate in both the initiation
step and the repetitive chain elongation steps. At this point, all glycosidic
bonds in the polysaccharide are in the α-1→4 configuration, so that this stage of
nascent glycogen resembles amylose. The enzyme responsible for the initiation
and extension of the linear polymer is glycogen synthase.
It is interesting to note that the carbon 1 of each glucose subunit is in the
α-configuration throughout, both in UDP-glucose, and in glycogen. What does
this tell us about the mechanism of the reaction? Nucleophilic substitutions
can occur either synchronously or asynchronously. In the first case, which is
called the SN 2 mechanism, one substituent leaves as the other arrives; each of
them holds on with ‘half a bond’ to the transition state.
As a result, the new substituent will occupy a position opposite to the
old one. With asymmetric C atoms, this gives rise to the so-called Walden
inversion. Since no such inversion occurs in glycogen chain elongation (no
conversion from α- to β-configuration; Figure 8.3), it follows that this cannot be
the correct mechanism. Instead, the first substituent (UDP) leaves, taking both
bond electrons with it. The carbon remains as a carbocation with three ligands
only. The enzyme then directs the new substituent—the terminal glucose of
the growing glycogen chain—to attack from the same side the UDP left. This is
therefore an SN 1 reaction mechanism.3

2 Whereever pyrophosphate is released, it is subsequently cleaved to two phosphate ions by

pyrophosphatase. This cleavage is exergonic and keeps the concentration of pyrophosphate


low, which in turn makes the first reaction more exergonic. Release of pyrophosphate therefore
provides a stronger driving force to a reaction than release of monophosphate.
3 With non-enzymatic S 1 reactions, we would expect racemization to occur at this stage, as
N
the new substituent could attack the planar transition state with equal ease from either side.
Enzymes, however, can impose a defined route of attack.
100
OH
a) O P O UTP O c)
O C HN
O
O O O O
OH N

OH OH O P O P O P O C
O UDP-glc UDP-glc UDP-glc UDP-glc UDP-glc
OH O O O
CH2OH
Glucose-6-P O

OH O OH OH

OH O P O
O
HO O O O O O O-Tyr Glycogenin
OH OH
Glucose-1-P CH2OH
HN
O
UDP UDP UDP UDP UDP
OH O O O N
OH O P O P O C
O
2 Pi P-Pi OH OH O

UDPglucose OH OH

b) CH2OH
HN
d)
O CH2OH
OH O O O N
OH O P O P O C
HO Tyr Glycogenin O O O O O O O O-Tyr Glycogenin
O
OH OH O

OH OH

HN
HO O O
O
O O O CH2OH
N

8 Glycogen metabolism
HO P O P O C
O
O CH2
O O OH
OH O HO O O O O O-Tyr Glycogenin
Tyr Glycogenin
OH OH OH
UDP

Figure 8.2 Reactions in glycogen synthesis (1). a: Activation of glucose. Glucose-6-phosphate is converted to glucose-1-phosphate
by phosphoglucomutase. Glucose-1-phosphate uridylyltransferase converts glucose-1-phosphate to UDP-glucose. Pyrophosphate
released is cleaved by pyrophosphatase. b: 1 molecule of UDP- glucose reacts with glycogenin. c: Glycogen synthase linearly
polymerizes glucose residues, again using UDP-glucose as a substrate. d: Branching enzyme transfers the end of a growing chain
onto the C6 atom of an internal glucose residue. Both C4 ends are subsequently extended by glycogen synthase until branching
repeats.
8.2 Glycogen degradation 101

8.1.3 Branching enzyme


Branching enzyme cuts a string of glucose residues from a growing end and
grafts it onto the sixth C atom of a glucose residue within a chain (Figure 8.2).
Branching can be repeated, such that the distance between adjacent branch
points will be 6-8 glucose residues, and branches will carry other branches in
turn. What is the purpose of this? Branching increases the number of free ends
for attachment or—during degradation—removal of single glucose residues.
The higher number of branches in glycogen relative to amylose and amylopectin
is in line with the fact that animals have a higher metabolic turnover than
plants.

8.2 Glycogen degradation


Two enzymes collaborate in glycogen degradation: Phosphorylase and de-
branching enzyme. All glucose subunits that are joined by α-1→4-glycosidic
bonds—that is, those in the straight segments—will be released by glycogen
phosphorylase. While the most common way of cleaving glycosidic bonds in
metabolism consists simply in hydrolysis, phosphorylase uses phosphate ions
instead of water to cleave these bonds (Figure 8.4). While this is not an ex-
ergonic reaction under standard conditions, it works here because the free
concentration of glucose-1-phosphate in the cell is low. Glucose-1-phosphate
is converted back to glucose-6-phosphate. In the liver, which stores glycogen
for the benefit of the entire body, the lion’s share will be dephosphorylated by
glucose-6-phosphatase and released into the circulation. As pointed out above,
the same may happen in muscle; however, muscle certainly uses glycogen to a
large extent for its own keepup, and therefore glucose-6-phosphate will often
be funneled straight into glycolysis.
Glycogen phosphorylase only degrades the chain ends to within 4 residues
of a branching point. Then, debranching enzyme takes over and transplants the
stub to another free end. However, it leaves behind a single residue attached
by a α-1→6-glycosidic bond, which it subsequently cleaves by hydrolysis and
releases as glucose (Figure 8.4b).

8.3 Glycogen storage diseases


While the above reactions should account for the complete degradation of
glycogen (except for the very last glucose residue attached to glcyogenin), there
is more than meets the eye. This is evident from the fact that a genetic defect
in an apparently unrelated enzyme—lysosomal maltase,4 which cleaves the α-
1→4-glycosidic bond between two glucose residues—causes the accumulation
4 The reaction is the same as catalyzed by the brush border maltase in the degradation of

amylose; however, the lysosomal enzyme is a separate entity and has an acidic pH optimum, in
keeping with the acidic environment inside the lysosomes.
102 8 Glycogen metabolism

CH2OH
a) O
CH2OH
OH
O
O Glycogen
O OH
OH
CH2OH
CH2OH O Glycogen
O O
H O OH
OH
OH H
OH O UDP OH
OH O UDP
CH2OH OH

OH

CH2OH O Glycogen
O
O OH

OH

OH H +UDP
OH

CH2OH CH2OH
b)
O CH2OH O
H
OH O OH
+
OH O UDP OH C H O Glycogen
O
OH OH OH

OH
O UDP

CH2OH CH2OH

O H O

OH OH

OH O Glycogen
O
OH OH

Figure 8.3 Reaction mechanism of glycogen synthase. a: A SN 2 mechanism should


result in a Walden inversion, converting the α-glycosidic bond in UDP-glucose to a β-
glycosidic bond, which is not observed. b: A SN 1 mechanism is compatible with the
retention of the α-configuration.

of glycogen in all kinds of tissues, with particularly detrimental consequences in


heart muscle. The accumulation of glycogen pudding inside the cell interferes
with cell motility and therefore with the contraction of the heart, and heart
failure leads to death. The condition is known as Pompe’s disease.
Other enzymes may be deficient as well. A deficiency of phosphorylase in
the muscle is known as McArdle’s disease; patients suffer from rapid exhaus-
tion and muscle pain during exertion. A defect of glucose-6-phosphatase is
mostly manifest in the liver and kidney. It will affect the release of glucose
from glucose-6-phosphate formed by both glycogen degradation and gluconeo-
genesis, which in turn leads to critically low blood glucose (hypoglycemia).
8.4 Regulation of glycogen metabolism 103

a) Pi
glc–glc–glc–glc–glc–glc–glc–glc

glc-1-P
Pi
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc

glc-1-P

b)
glc–glc–glc–glc

glc–glc–glc–glc–glc–glc–glc–glc–glc–glc

glc

glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc

glc

glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc

Figure 8.4 Degradation of glycogen. a: Glycogen phosphorylase cleaves α-1→4-


glycosidic bonds using phosphate ions to yield glucose-1-phosphate. Phosphorylase
stops 4 glucose residues shy of a branching point. b: Debranching enzyme cleaves all
but one residue off a branch and adds them to another free end, which can then again be
degraded by phosphorylase. The remaining single residue (held by a α-1→6-glycosidic
bond) is removed by hydrolysis.

8.4 Regulation of glycogen metabolism


We have seen before that phosphofructokinase and the complementary enzyme
fructose-1,6-bisphosphatase are regulated by both intracellular and extracellu-
lar signals. The same applies to the enzymes involved in glycogen metabolism.
The allosteric regulatory effects by ATP, AMP and glucose-6-phosphate (Fig-
ure 8.5a) make sense: Depletion of ATP would be an excellent reason to regener-
ate it by tapping into the glucose store. On the other hand, glucose-6-phosphate
will be plentyful when glucose itself is abundant, so it should promote glycogen
synthesis rather than breakdown. Hormonal control (Figure 8.5b) is similar to
that of gluconeogenesis (section 7.3): Protein kinase A decreases glycogen syn-
thesis via direct phosphorylation of glycogen synthase. Glycogen breakdown
is stimulated by phosphorylation of a dedicated enzyme, phosphorylase kinase,
which then in turn phosphorylates glycogen phosphorylase. Note that glycogen
104 8 Glycogen metabolism

Figure 8.5 a) Glycogen


Regulation of
glycogen metabo-
lism. a: Allosteric
+ Glc-6-P -
control of glycogen
synthase and of glycogen phosphorylase
synthase ATP -
phosphorylase. b:
Hormonal control.
AMP +
See text for details.
UDP-glucose

Glucose-1-P

b) Epinephrin, glucagon Insulin

+
+

ATP cAMP AMP


+

Protein kinase A

Glycogen Glycogen
synthase (active) synthase–P (inactive)

Phosphorylase Phosphorylase
kinase (inactive) kinase–P (active)

Phosphorylase-P Phosphorylase
(active) (inactive)

synthase and phosphorylase respond in opposite ways to phosphorylation: The


first one is inactivated, the second activated.
There are regulatory differences between glycogen phosphorylase in muscle
and liver: Glucose inhibits the liver enzyme but not the muscle enzyme, and
Ca++ stimulates the muscle enzyme but not the liver enzyme. Recall that Ca++
is also the trigger for muscle contraction, so it seems that the simultaneous
stimulation of glycogen breakdown occurs in anticipation of increased ATP
requirement. This is an example of the usefulness of isozymes, that is enzymes
that catalyze the same reaction yet are different molecules, and therefore can
possess different regulatory adaptations.
Chapter 9

The hexose monophosphate shunt

We are (finally) nearly done with glucose-6-phosphate. Figure 9.1 summarizes


the various pathways that it is part of, and I hope that by now you recognize
most of them. The final one of these pathways to be covered in this class is
the hexose monophosphate shunt. Since both pentoses and hexoses—as well
as trioses, tetraoses, and heptoses—occur in it, this pathway is sometimes also
referred to as the ‘pentose phosphate pathway’.
A single passage of glucose-6-phosphate through the hexose monophos-
phate shunt oxidizes it to the C5-sugar ribulose-5-phosphate, releasing one
molecule of CO2 . In the process, two molecules of hydrogen are transferred to
NADP+ , yielding NADPH. The ribulose-5-phosphate can be turned into ribose-5-
phosphate and then used for the biosynthesis of nucleotides. Alternatively, it
can be fully oxidized to yield more CO2 and NADPH (Figure 9.2). The hexose
monophosphate shunt therefore provides a second means for complete degra-
dation of glucose to CO2 , apart from the glycolysis / TCA pathway we have
seen before. However, the purpose of this second oxidative pathway consists
not in the regeneration of ATP but in the formation of NADPH. This coenzyme
is required in many biosynthetic reactions, some of which we will consider be-
low. Glycolysis and TCA don’t fill this need, because all hydrogen they abstract
accumulates as NADH or FADH2 .1
1 Note, however, that there are some auxiliary pathways that can convert NADH to NADPH; one

of them is discussed in section 7.7.

105
106 9 The hexose monophosphate shunt

Glucose and other sugars as elements of polysaccharides, glycolipids,


glycoproteins

Glycogen UDP-Glucose

Glucose-1-phosphate Ribulose-5-P + CO2 + 2 NADPH

Glucose Glucose-6-phosphate 6-P-Gluconolactone

Fructose-6-phosphate

Pyruvate

6 CO2 + 6H2O

Figure 9.1 Metabolic fates of glucose-6-phosphate. The hexose monophosphate


shunt is indicated by underlining.

Dehydrogenation of pyruvate and the TCA occur in the mitochondria, which


is useful because the NADH generated is then fed into the respiratory chain. In
contrast, the hexose monophosphate shunt occurs entirely in the cytoplasm.
This is in keeping with the fact that most of the biosynthetic reactions involving
NADPH also occur in the cytoplasm (or in the ER, which is still outside the
mitochondrion).

9.1 Reactions in the hexose monophosphate shunt

The reactions occurring in the hexose monophosphate shunt can be divided


into two phases (Figure 9.2a): (1) Oxidation of glucose-6-phosphate to ribu-
lose-5-phosphate, and (2) regeneration of glucose-6-phosphate from ribulose-5-
phosphate.
The two phases may operate at the same time, or independently from each
other. While glucose oxidation is irreversible, the interconversion of glucose-
6-phosphate to pentose phosphates is reversible (Figure 9.2b). With a typical
diet, reasonably rich in starch, the net flow of sugar conversion will be from
hexoses to pentoses. However, when eating meat only, our intake of ribose in
the form of RNA will be a very significant fraction of the total carbohydrates,
and the net flow in the hexose monophosphate shunt will likely go the other
way (Figure 9.2b, bottom).
9.1 Reactions in the hexose monophosphate shunt 107

a) Glucose-6-P biosynthesis
of nucleotides and
nucleic acids

Regenerate 5 2 NADP+
Glucose-6-P 2 NADPH
from 6 Ribulose-5-P CO2

Ribulose-5-P Ribose-5-P

b) CO2

Pentose-P Glucose-P NADPH

Pentose-P Glucose-P NADPH

Pentose-P Glucose-P NADPH

Figure 9.2 Overview of the hexose monophosphate shunt. a: Glucose-6-phosphate


is oxidized and decarboxylated to ribulose-5-phosphate. The hydrogen abstracted is
transferred to NADP+. The ribulose-5-phosphate can be converted back to a propor-
tionally dimished amount of glucose-6-phosphate, or it can be diverted for the purpose
of nucleotide synthesis. b: The net flow of metabolites through the hexose monophos-
phate shunt may vary depending on the metabolic situation.

9.1.1 Reactions in the oxidative stage


Three enzymes are required for the oxidative phase (Figure 9.3):
1. Glucose-6-phosphate dehydrogenase reduces one molecule of NADP+ to
NADPH and produces 6-phosphogluconolactone.
2. Gluconolactonase cleaves the internal ester bond and produces 6- phos-
phogluconate.
3. 6-Phosphogluconate dehydrogenase reduces another molecule of NADP+
and decarboxylates 6-phosphogluconate to the pentose ribulose-5-phosphate.
After completion of the oxidative phase, NADPH generation is over, and
everything that remains is juggling sugars in order to regenerate hexoses from
pentoses.

9.1.2 Reactions in the sugar juggling phase


It is important to note that the formation of hexoses from pentoses is not
stoichiometric for the sugar molecules. Instead, it is stoichiometric for the
carbons within them: 6 molecules of the C5 compound ribulose-5-phosphate
are converted to 5 molecules of the C6 compound glucose-6-phosphate.
108 9 The hexose monophosphate shunt

OH OH

O P O Glucose-6-P NADPH+H+ O P O 6-P-Glucono-


O C O C lactone
NADP+
O O

OH OH O

OH OH 1 OH

OH OH

H 2O
2
O
HO
Ribulose-5-P NADPH+H+
OH OH
NADP+ HO 6-P-Gluconate
O

OH OH
3
OH O OH O
CO2
Nucleotides O P OH O P OH

O O

Figure 9.3 The oxidative stage of the hexose monophosphate shunt. Enzymes:
1: glucose-6- phosphate dehydrogenase; 2: 6-phosphoglucolactonase; 3: 6-
phosphogluconate dehydrogenase.

The reactions in the second phase of the hexose monophosphate shunt are
depicted in Figure 9.4:
1. Two molecules of ribulose-5-phosphate are converted to xylulose-5- phos-
phate and to ribose-5-phosphate by ribulose-5-phosphate epimerase and ribu-
lose-5-phosphate isomerase, respectively.
2. Transketolase transfers a C2 unit from the xylulose-5-phosphate to the
ribose-5-phosphate, yielding glyceraldehyde-3-phosphate and the C7 sugar se-
duheptulose-7-phosphate.
3. Transaldolase transfers a C3 unit from the seduheptulose-7-phosphate
back to glyceraldehyde-3-phosphate, yielding fructose-6-phosphate and the C4
sugar erythrose-4-phosphate. The fructose-6-phosphate may enter glycolysis,
or may be converted back to glucose-6-phosphate by phosphohexose isomerase.
4. Transketolase transfers a C2 unit from another molecule of xylulose-5-
phosphate to erythrose-4-phosphate. This yields a second molecule of fructose-
6-phosphate and again glyceraldehyde-3-phosphate. Both may enter glycolysis
or be converted back to glucose-6-phosphate.
The conversion of glyceraldehyde-3-phosphate back to glucose-6-phosphate
via fructose-1,6-bisphosphate utilizes the enzymes from gluconeogenesis and
glycolysis that you so fondly remember from the previous chapters. Recall
that aldolase needs two molecules of triose phosphate to form one molecule of
fructose-1,6- bisphosphate. This results in an overall stoichiometry of 2.5 mo-
lecules of hexose per 3 molecules of pentose, or five hexoses per six pentoses.
9.1 Reactions in the hexose monophosphate shunt 109

OH

OH OH O

O O HO

OH HO TK O OH
RE
OH OH OH OH

O P O P O P O P

C5 C5 C3 TA C6
OH

O OH

OH O HO O

O OH OH O HO

OH OH OH OH OH
RI
OH OH OH OH OH

O P O P O P O P O P

C5 C5 C7 C4 C6

TK
O P

OH OH O

O O HO

OH HO O OH
RE GN
OH OH OH OH

O P O P O P O P

C5 C5 C3 0.5 C6

Figure 9.4 The glucose-6-phosphate regeneration phase. Enzymes: RE, ribulose-


5-phosphate epimerase; RI, ribulose-5-phosphate isomerase; TK: Transketolase; TA:
Transaldolase; GN, Enzymes from gluconeogenesis. Braces indicate the C2 and C3 units
transferred by transketolase and transaldolase, respectively.

The juggling of sugar chain length depicted in Figure 9.4 is brought about
by two enzymes, transaldolase and transketolase. While the sugar substrates
they act upon appear to be quite varied at first glance, they fall into just two
structural classes, the members of each of which differ only in chain length
(Figure 9.5). The two enzymes only interact with the topmost parts of these
sugar molecule, so that the chain length of the remainder doesn’t enter into
the picture and does not create a need for separate enzymes. The basic idea of
chain length variation is that transketolase always transfers two-carbon units,
whereas transaldolase always transfers three-carbon units. Sequential reaction
110 9 The hexose monophosphate shunt

Xylulose-5-P Fructose-6-P Seduheptulose-7-P

OH OH OH OH

O O O O

OH HO HO HO

OH OH OH OH Ketoses
O P O P OH OH

Ribulose-5-P O P OH
O
O P
OH O

OH OH O
Aldoses
OH OH OH

O P O P O P

Ribose-5-P Erythrose-4-P Glyceraldehyde-3-P

Figure 9.5 The intermediates of the glucose-6-phosphate regeneration phase form


two homologous series of ketoses and aldoses, respectively. Transketolase and transal-
dolase cleave C2 and C3 units, respectively, from the ketoses and graft them onto the
aldehyde group of the aldoses.

of the two enzymes will therefore lead to a net change of the substrate chain
length by one carbon.

9.2 Mechanisms of transketolase and transaldolase


The mechanisms of transketolase and transaldolase are depicted in Figure 9.6.
Transketolase employs the coenzyme thiamine pyrophosphate, which we en-
countered before in the pyruvate dehydrogenase E1 enzyme. As in the pyruvate
dehydrogenase reaction, its main function is to provide a carbanion, which
reacts with a carbonyl group and cleaves the adjacent C−C bond, yielding a
covalent bond between the coenzyme and the substrate. However, here the sim-
ilarity ends. In the second part of the reaction, another aldose substrate enters
and carries the transiently coenzyme-bound C2 subunit away. The mechanism
of the second half-reaction is exactly the reversal of the first part.
Transaldolase also forms a covalent intermediate with the fragment of the
sugar molecule it transfers, and once more the carbonyl bond serves as the
point of attack for cleavage. However, transaldolase cleaves the bond after the
second carbon atoms, resulting in the transfer of a C3 unit. The two stages of
the reaction are again reversals of each other, with the exception that sugar
substrates of different length will participate.
Considering their mechanisms, it makes sense that both the transketolase
and the transaldolase reactions are readily reversible. So are the isomerase
reactions that interconvert the various pentose phosphates. This pathway
9.3 Why do we need both NADH and NADPH? 111

therefore can go either way and bring about the interconversion of pentoses,
hexoses and sugars of other lengths as needed.

9.3 Why do we need both NADH and NADPH?


Why NADH and NADPHdifferent roles of is NADPH needed in addition to NADH?
The two coenzymes only differ by one phosphate group, and that is far away
from where the action is: The redox-active group is the nicotinamide moiety,
whereas the adenosine moiety is the one that carries the extra phosphate in
NADP (Figure 3.7). While the phosphate group does not make any difference
to the redox chemistry performed by the two coenzymes,2 it enables them to
interact with different sets of enzymes. Consider that all enzymes that consume
or regenerate NAD+ will share the same pool of the coenzyme, and the reaction
equilibria of all of them will be affected by the same ratio of oxidized over
+
reduced form ([NAD ]/[NADH]).
While NADP completely resembles NAD in its redox chemistry, the extra
phosphate group allows NADP to interact with a different set of enzymes,
which does not intersect with the NAD-specific set. Therefore, because the
coenzymes participate in different sets of equilibria, they can themselves be
maintained in different redox states. To use a simile: The two coenzymes are
like to different currencies – both are money, but it is possible to tune the cost
of borrowing to different economic conditions. Inside the cell, NAD is mostly
oxidized. The ready availability of NAD+ will help to speed up the oxidative
reactions in the TCA and glycolysis. In contrast, NADP is mainly found in the
reduced state, which will promote reductive reactions in biosynthesis.
Apart from the reaction rates, the free energy (∆G) of the redox reactions
will also be affected by the “choice” of either NAD or NADP as the cosubstrate.
Using values of 0.001 for the ratio of [NADH]/[NAD+ ] and of 100 for [NADPH]/[NADP+ ],
and assuming ∆G0 to be the same for both, this difference works out to about
30 kJ/mol.3

9.4 Alternative sources of NADPH


Not all cells that require large amounts of NADPH have a high activity of the
hexose monophosphate shunt. Some alternative mechanisms that can provide
NADPH are:
1. The combination of pyruvate carboxylase, malate dehydrogenase, and
malic enzyme, which generates NADPH from NADH at the expense of ATP
hydrolysis (see section 12.1).
2 If we use the same concentrations of reduced and oxidized forms in vitro, there is practically

no difference in their redox potentials, i.e. they have the same standard redox potentials.
3 This ∆G is similar to that provided by the hydrolysis of ATP to ADP. For the cytosol, this

sounds about right (see section 9.4 below). It is probably less in mitochondria, where NADH and
NADPH are distinguished only by the energy of a single proton transfer (via transhydrogenase).
112 9 The hexose monophosphate shunt

R
a)
S H2C OH
H3C
R
+ C O H
N
S H2C OH R HC OH
H3C
+ C
N C O H+ HC OH

R HO CH R HC OH

HC OH
HC OH
S H2C OH
H2C O P H3C
OH C O P
+ C H2
N

R
H2C OH
R +
O
+ H+
H

S H2C OH HC O HO CH
H3C
+ C OH HC OH HC OH
N

R O CH HC O HC OH HC OH
H
HC OH HC OH HC OH HC OH

H2C O P H2C O P H2C O P H2C O P

b) H2C OH H2C OH H2C OH


+
E Lys NH2 C O E Lys N C E Lys N C
H H
HO CH HO CH HO CH

HC OH HC O H HC O

HC OH HC OH HC OH

HC OH H 2O HC OH
H+ HC OH

H2C O P H2C O P H2C O P

H2C OH H2C OH H2C OH


E Lys NH2
+
E Lys N C E Lys N C O
H H
HO CH HO CH HO CH
+
H
HC O HC O H HC OH

HC OH HC OH HC OH
H 2O
H2C O P H2C O P C O P
H2

Figure 9.6 Mechanisms of transketolase and transaldolase. a: Transketolase. Only


the thiazole ring of the thiamine pyrophosphate is shown. The formation of the carban-
ion is as described before for pyruvate dehydrogenase (see Figure 5.4). b: Transaldolase.
The –N=C– group that links the substrate to the enzyme in the covalent intermediate is
a ketimine or Schiff base.
9.5 Uses of NADPH 113

2. NADP+ -dependendent isocitrate dehydrogenase, which occurs alongside


the NAD+ -dependent enzyme in mitochondria (see chapter 5) but also in the
cytosol. This mechanism seems to be particularly important for fatty acid
synthesis in the liver and the fat tissue (see chapter 10).

9.5 Uses of NADPH


So, where does the NADPH generated in the hexose monophosphate shunt go?
Here are several destinations:
1. Synthesis of fatty acids,
2. Synthesis of cholesterol,
3. Fixation of ammonia by glutamate dehydrogenase,
4. Oxidative metabolism by cytochrome P450 enzymes:
(a) Generation of catecholamine mediators (dopamine, epinephrine and
norepinephrine),
(b) Drug metabolism,
5. Generation of nitric oxide and reactive oxygen species by phagocytes,
6. Scavenging of reactive oxygen species that form as byproducts of oxygen
transport and of the respiratory chain.
Topics 1–3 are going to be covered in detail in subsequent chapters. Here,
we will have a brief look at topics 4–6.

9.5.1 Cytochrome P450 enzymes and drug metabolism


Cytochrome P450 enzymes split molecular oxygen (O2 ) and use NADPH to re-
duce one of the atoms to water. The other oxygen atom is retained in a highly
reactive form and utilized to force some kind of reaction on an intrinsically
reluctant substrate. This is a large class of enzymes that serves a wide vari-
ety of metabolic tasks; examples are hydroxylations and epoxydations in the
synthesis of catecholamine and steroid hormones. Cytochrome P450 enzymes
also have a prominent place in the metabolic elimination of drugs. While there
are multiple types of reactions in drug metabolism, one important example
is the hydroxylation of aliphatic or aromatic moieties in drug molecules. The
hydroxyl group introduced can then be conjugated with hydrophilic groups,
which facilitates elimination in the urine (Figure 9.7).

9.5.2 Production of reactive oxygen species


While cytochrome P450 enzymes utilize reactive oxygen in a controlled, con-
tained fashion, there are other enzymes that generate reactive oxygen species
to release them within or outside the cell, for the purpose of killing pathogenic
microbes. This happens in macrophages and neutrophile granulocytes. Reac-
tive oxygen species include the superoxide anion ( · O−O – ), hydrogen peroxide
(H2 O2 ), and hypochlorite (H−O−Cl; this is also the active constituent of bleach).
114 9 The hexose monophosphate shunt

a) Hydrophilic drug molecule Hydrophobic drug molecule

Liver

Kidney More hydrophilic metabolite

Urine

b)
NADP+
O O
H H
C2H5 N NADPH+H+ C2H5 N
O O
N HO N
H H
O O2 H2O O

O
O O H O O
C C2H5 N C
O O O
OH O N OH
H
OH O UDP OH O UDP
OH OH

Figure 9.7 Role of NADPH in drug metabolism. a: Overview: Drug metabolism occurs
in the liver and often serves to convert hydrophobic drug molecules to hydrophilic
metabolites, which facilitates excretion in the urine. b: Example: NADPH- dependent
hydroxylation of the drug phenobarbital. Hydroxylation is followed by conjugation
with glucuronic acid, which is derived from UDP-glucuronic acid.

The enzyme reactions involved in their formation are summarized in Figure


9.8a. NADPH oxidase is responsible for the initial formation of superoxide,
from which the other intermediates are derived.

The enzymes are contained in small vesicles called peroxisomes, which will
fuse with phagosomes that contain ingested microbes. The reactive oxygen
species (ROS) generated have an important role in the destruction of the mi-
crobes. This is shown by the fact that enzyme defects in the formation of ROS
cause severe immunodeficiencies, in particular with respect to bacterial infec-
tions. ROS also have an important share in the tissue destruction that occurs
in inflammation.
9.6 Glucose-6-phosphate dehydrogenase deficiency 115

a) 2 O2 + NADPH 2 O2- + NADP+ + H+


NADPH oxidase

2 O 2- + 2 H + H 2O 2 + O 2
Superoxide dismutase

H2O2 + Cl- H2O + O-Cl-


Myeloperoxidase

b)

ROS

Figure 9.8 Generation of reactive oxygen species (ROS) in granulocytes. a: Sum-


mary of the enzymatic reactions involved. b: Release of enzymes into a phagosome
containing an ingested bacterium generates ROS and kills the bacterium.

9.5.3 Scavenging of reactive oxygen species


Where not needed for immune defense, reactive oxygen species are harmful
rather than useful. Nevertheless, some ROS always form as byproducts of res-
piration, or even of oxygen transport in erythrocytes, since binding to heme
offers oxygen an opportunity to steal an electron. This toxicity is controlled
by the presence of glutathione (Figure 9.9a). An important step in the detox-
ification of reactive oxygen is the reduction of hydrogen peroxide (H2 O2 ) to
water by glutathione peroxidase (Figure 9.9b). Glutathione is then regenerated
by glutathione reductase at the expense of NADPH.

9.6 Glucose-6-phosphate dehydrogenase deficiency


Glucose-6-phosphate dehydrogenase is an essential enzyme, and complete lack
of it is not compatible with life. However, there are hereditary mutations
116 9 The hexose monophosphate shunt

O
a)
O O

N SH
H
NH

O O

O NH2

O O

O O O O

N S S N
H H
NH N
H
O O O O

O NH2 NH2 O

b) H2O2 2 G-SH NADP+

Glutathione Glutathione
peroxidase reductase

2 H2O G-SS-G NADPH+H+

c) O

O
HN

2 GSH H3C N NH H 2O 2 2 GSH

Glutathione
spontaneous spontaneous
peroxidase
O

GSSG OH O2 GSSG
HN

H3C N NH2

Figure 9.9 The role of glutathione in scavenging of reactive oxygen species, and
the mechanism of glutathione depletion in favism. a: Glutathione in its reduced and
oxidized states. b: Glutathione peroxidase oxidizes glutathione and reduces hydrogen
peroxide to water. Reduced glutathione is regenerated using NADPH by glutathione
reductase. c: Isouramil is one of several similar compounds found in broad beans.
It cycles between reduced and oxidized states; each cycle oxidizes four molecules of
glutathione.
9.6 Glucose-6-phosphate dehydrogenase deficiency 117

characterized by reduced enzyme activity. The defect becomes manifest mostly


in erythrocytes. This is related to the fact that erythrocytes have no protein
synthesis, which has two consequences:
1. All protein molecules, including enzymes, have to last for the entire
lifetime of the cell, which is normally 120 days. In contrast, in nucleated cells
the lifetime of an enzyme molecule is typically in the range of hours to a few
days. Reduced intrinsic stability of the enzyme may be masked by fast turnover
in nucleated cells but become evident in red cells.
2. In nucleated cells, there typically is a mechanism that adjusts enzyme
expression to metabolic needs; reduced specific enzyme activity may therefore
be partially compensated by increased expression. No such mechanism exists
in red cells.
If the enzyme defect becomes manifest, the reduced throughput of the hex-
ose monophosphate shunt will limit the regeneration of glutathione. Therefore,
oxidative stress will result in glutathione depletion, which may lead to cell dam-
age. In most people, the glucose-6-phosphate dehydrogenase defect does not
show up until they ingest some drug or food constituent that places increased
oxidative stress on the red cells. The classical trigger is the broad bean, vicia
fava, and the condition is therefore known as favism. The broad bean contains
several pyrimidine bases that undergo redox reactions with both oxygen and
glutathione. This leads to a catalytic cycle, in which each molecule of base oxi-
dizes many molecules of glutathione, which in turn is reduced at the expense
of NADPH. This is illustrated for one of these bases, isouramil, in Figure 9.9c.
Ingestion of broad beans results in consumption of NADPH in both healthy
individuals and those afflicted by glucose-6-phosphate dehydrogenase defi-
ciency. However, while healthy individuals can compensate this, NADPH be-
comes depleted in those with the enzyme defect. The red cells will then suffer
destruction (= hemolysis) at the hands of unscavenged reactive oxygen species.
Hemolytic crises can also be caused by certain drugs, including anti-infectious
agents such as sulfonamides and metabolites of the anti-malarial drug pri-
maquine, which can undergo cyclical oxidation and reduction just like isouramil.
To what extent this effect is involved in the therapeutic effect of primaquine in
malaria is unclear. So, it is very important to watch out for glucose-6-phosphate
dehydrogenase deficiency in patients to be treated with one of these drugs.
Like sickle cell anemia and other hemoglobinopathias, glucose-6-phosphate
dehydrogenase deficiency is particularly common in areas with widespread
occurrence of malaria. Like sickle cell anemia, it supposedly affords some pro-
tection against malaria; the biochemical mechanism of this protection remains
unsettled.
Chapter 10

Triacylglycerol metabolism

Various types of lipids occur in the human body: (1) Triacylglycerol, (2) choles-
terol, and (3) polar lipids, which include phospholipids, glycolipids and sphin-
golipids.
While polar lipids and cholesterol are found in the cell membranes of ev-
ery cell, triacylglycerol is essentially confined to fat tissue, which stores and
releases it, and to the cells in the intestine and the liver that synthesize and
degrade it.1 Yet, triacylglycerol is the most abundant lipid species, and the only
one with an important role in energy metabolism. We will therefore here focus
on triacylglycerol. Cholesterol, which is not important in energy metabolism,
will be covered in a separate chapter as well because of its medical importance.
Triacylglycerol occurs in human metabolism in two roles: (1) As a foodstuff.
A significant fraction of our caloric intake is triacylglycerol. (2) As an endoge-
nous store of metabolic energy. This storage can be replenished from dietary
fat, or by endogenous synthesis of fat from carbohydrates or proteins.
The amount of energy per gram of tissue is far higher in fat than in any
other tissue, for two reasons: (1) One gram of triacylglycerol itself contains
roughly twice as many calories as one gram of carbohydrates or protein. This is
because triacylglycerol contains much less oxygen than carbohydrates, in which
oxygen contributes half the mass but essentially no metabolic energy. Similarly,
1 There is also some intracellular storage of triacylglycerol, for example in the muscle cells,

but it is quantitatively insignificant relative to adipose tissue.

118
Triacylglycerol metabolism 119

H2 H H2 OH
a) C C C
O O O
OH OH OH O

O O O CH2
Glycerol H2C

CH2

H2C

CH2

H2C

CH2

H2C

CH2

H2C

CH2

H2C

CH2
Palmitic acid
H2C
(hexadecanoic acid)
CH3

b) ketone bodies

5 6 ADP ATP
3
fatty acids acetyl-CoA CO2 + H2O
4 7
1 2
Triacyl-
glycerol

glycerol

glucose pyruvate

Figure 10.1 Overview of triacylglycerol metabolism. a: Structure of tripalmitin, an


example species of triacylglycerol. Three molecules of fatty acid—here: palmitic acid—
are esterified to the three hydroxyl groups of glycerol. b: Pathways: Triacylglycerol
breakdown (1) and synthesis (2), β-oxidation (3), fatty acid synthesis (4), ketone body
synthesis (5) and utilization (6), TCA and respiratory chain (7). The dashed line repre-
sents the “Rubicon” between carbohydrates and fat: Once pyruvate is decarboxylated,
the remaining carbon cannot be turned back to glucose anymore.
120 10 Triacylglycerol metabolism

protein contains oxygen, nitrogen and sulfur and is similar in energy content
per dry weight to carbohydrates. (2) Triacylglycerol in fat cells coalesces to
droplets that are entirely free of water. In contrast, protein and carbohydrates,
including glycogen, always remain hydrated.
Because of this efficiency of storage, it makes sense that most of the excess
glucose or protein is converted to fat, while only a limited fraction is stored as
glycogen. There is, however, one limitation to the usefulness of triacylglycerol:
Once carbohydrate or protein carbon has been converted to fat, it can go back
and forth between fat, fatty acids, acetyl-CoA, and ketone bodies, but it is no
longer available for the regeneration of glucose (Figure 10.1b). Therefore, when
starving, we will always have to degrade some protein along with fat to keep
up a minimum supply of glucose, and therefore starving people from day zero
will not only deplete their fat stores but also their muscle tissue.

10.1 Utilization of dietary triacylglycerol


10.1.1 Solubilization and digestion
Digestion of triacylglycerol requires two components: (1) Bile acids. These
act as detergents and solubilize the chunks or droplets of ingested fat into
small micelles and in this way render the triacylglycerol accessible to enzy-
matic cleavage. One of the major bile acids, cholic acid, is shown in Figure 11.1.
(2) Pancreatic lipase. This esterase hydrolyses triacylglycerol to 2-monoacyl-
glycerol and two molecules of free fatty acids.
The enzyme can act upon the triacylglycerol molecules only at the surface of
the micelles. Dispersal of the lipid particles by bile acids is therefore necessary
for hydrolysis. The fatty acids released by lipase act as detergents themselves
and will aid in the solubilization of remaining fat (Figure 10.2).2
After solubilization and hydrolysis by the lipase enzymes, the monoacyl-
glycerol and the free fatty acids are taken up by the intestinal epithelial cells.
If either bile acids or pancreas lipase are lacking—due to malfunctions of liver
or pancreas, or to obstruction of the respective secretory ducts—fat malab-
sorption will occur, and fat will appear in the stool. In addition, fat-soluble
vitamins (A, D, E, K) will be carried down the drain along with the fat, and
vitamin deficiencies may ensue.

10.1.2 Formation of chylomicrons


After the products of triacylglycerol digestion—monoacylglycerol and fatty
acids—have been taken in the small intestine, the next thing to happen to them
is somewhat surprising: They are directly converted back to triacylglycerol
while still inside the intestinal epithelial cells. The newly formed fat is then
2 Curd soap is prepared simply by alkaline hydrolysis of fat; it consists of the sodium salts of

the fatty acids released.


10.1 Utilization of dietary triacylglycerol 121

a)

Fat

Monomeric
detergent

Detergent micelle Mixed micelle (solubilized fat)

b) c)
OH O OH
O O
O

micellar

CMC

monomer

total detergent concentration

Figure 10.2 Fat solubilization by detergents. a: Monomeric detergent molecules


penetrate the surface of a fat droplet and break it up into mixed micelles. b: Cleavage
of triacylglycerol by lipase yields 2-monoacylglycerol and free fatty acids, which act
as detergents and assist in the solubilization of remaining fat. c: Detergents may
exist in micellar and monomeric form. Below its critical micellar concentration (CMC),
a detergent dissolves completely as monomers; any excess above the CMC will form
micelles (cf. panel a, left). Bile acids have very high CMC values. They therefore have
high concentrations of available monomers and solubilize fat rapidly.
122 10 Triacylglycerol metabolism

combined with protein molecules called apolipoproteins into lipoprotein par-


ticles, such that the proteins form a hydrophilic shell around the lipid core
(Figure 10.3a). Some phospholipids are included as well and complete the
hydrophilic shell.
Apolipoproteins and lipoproteins occur in various subtypes (section 11.1).
The specific lipoprotein particles formed at this stage, the chylomicrons, are the
largest ones among all lipoproteins, with a molecular mass of up to 1010 Dalton,
a diameter up to 1 µm, and approximately 107 molecules of triacylglycerol. The
chylomicrons are released from the intestinal cells at the basolateral side. Since
they are so large, they cannot diffuse acrosse the capillary walls to reach the
blood stream—which small molecules can—but are instead drained into the
lymphatics. All lymph fluid is ultimately collected in the thoracic duct, which
in turn discharges into the main circulation, bypassing the liver. Therefore,
triacylglycerol is an exception from the general rule that all substances taken
up in the intestine are first passed through the liver.

10.1.3 Utilization of chylomicrons

The triacylglycerol contained in the chylomicrons can be utilized in various


ways: (1) It can be stored in fat cells, or (2) it can be utilized directly for the
purpose of ATP production by muscle cells and other tissues.
In both cases, the triacylglycerol in the chylomicrons again needs to get
across the barrier of the vascular endothelium. This is accomplished with
the help of lipoprotein lipase, an enzyme found at the endothelial surface. It
binds the chylomicrons and cleaves the triacylglycerol again to fatty acids and
glycerol. These are small molecules and can diffuse across the endothelial
barrier to reach the cells in the surrounding tissue. In fat cells, the fatty acids
are combined with glycerol yet again for storage (Figure 10.3b). Alternatively, in
other cells, they can be directly degraded to acetyl-CoA, which is then consumed
in the TCA and the respiratory chain. The remnants of chylomicrons, depleted
of most of the triacylglycerol, are captured by the liver,3 phagocytosed, and
degraded.
Why is the transport of triacylglycerol so complicated? It would seem easier
to do away with all these splitting and rejoining steps and just pour the fatty
acids into the blood. One reason is that fatty acids are toxic. As mentioned
before, they are detergents – and detergents solubilize cell membranes, that is
they cause cell destruction.4

3 Sooner or later, each particle in the blood will pay the liver a visit, even if it was initially

bypassed.
4 Interestingly, the surfaces of the intestinal cells withstand high concentrations of bile and

fatty acids! This phenomenon has not yet received appropriate scientific attention.
10.1 Utilization of dietary triacylglycerol 123

a) Fatty acids, 2-MAG

Fatty acids, 2-MAG Protein


ATP
ADP
intestinal
epithelium
Triacylglycerol Triacyl-
Apolipoproteins glycerol

Chylomicrons

Chylomicrons lymphatics

b)
Fat cell Triacylglycerol

Endothelial
cell

Fatty acids,
glycerol
Chylomicron

to Liver
Lipoprotein lipase

Figure 10.3 Intestinal uptake and post-processing of dietary fat. a: Left: Fatty acids
and monoacylglycerol are taken up by the intestinal cells, converted back to triacyl-
glycerol, and together with apolipoproteins packaged into chylomicrons. Right: Chy-
lomicrons (and other lipoproteins) are fat droplets surrounded by a protein shell. b:
Lipoprotein lipase binds chylomicrons and extracts and cleaves triacylglycerol. The
cleavage products are stored in fat cells (after being converted to triacylglycerol yet
again, as shown) or utilized by oxidative degradation.
124 10 Triacylglycerol metabolism

10.2 Utilization of fatty acids: β-Oxidation


As briefly mentioned before, the utilization of fatty acids occurs by way of
conversion to acetyl-CoA, which is accomplished in β-oxidation. This pathway
runs in the mitochondria, so the first task after cellular uptake of the fatty acid
molecule is to get it into the mitochondrion.

10.2.1 Mitochondrial transport of fatty acids


Fatty acids are initially activated to fatty acyl-CoA in the cytosol. This is also
the form in which they enter degradation by β-oxidation. However, during
transport, the CoA-moiety is transiently replaced by carnitine. The process is
outlined in Figure 10.4.
One unusual aspect of this transport process is that the energy of the thi-
oester bond in acyl-CoA seems to be sufficiently well preserved in the ester
bond of acylcarnitine to allow the exchange reaction to be reversed inside the
mitochondrion. Carboxyl esters bonds don’t usually have a sufficiently high
energy content for that. I once read a research paper on that topic, but it went
straight over my head. I will therefore have to owe you the explanation why
carnitine is special in this respect. On the bright side, you won’t have to know
it in the exam.

10.2.2 Reactions in β-oxidation


The term β-oxidation refers to the greek lettering of the carbons in organic
molecules: The α carbon is right next to a functional group, and the β carbon
is the second one (Figure 10.5a, top). It is at the second carbon from thioester,
then, where the action is in β-oxidation. Figure 10.5a gives a summary of the
reactions:
1. The fatty acyl-CoA molecule is first dehydrogenated between the α and
the β carbon atoms by acyl-CoA dehydrogenase. FAD accepts the hydrogen
abstracted and is reduced to FADH2 . This yields 2-trans-enoyl-CoA.
2. The trans double bond just created is hydrated—that is, water is added
across it—by enoyl-CoA hydratase, which yields hydroxyacyl-CoA. The α carbon
is now once more fully reduced.
3. The β-hydoxyl group is converted to a keto group by hydroxyacyl-CoA
dehydrogenase. NAD+ accepts the hydrogen. The product is β-ketoacyl-CoA.
4. Thiolase introduces a new molecule of coenzyme A to cleave the β-
ketoacyl-CoA, to release acetyl-CoA and a new, shortened acyl-CoA that enters
the next cycle of β-oxidation.
The process is repeated until the fatty acid is completely broken down. In
the case of even-numbered acyl chains, this will yield acetyl-CoA only, whereas
odd-numbered acyl chains will yield one molecule of propionyl-CoA in the final
step (Figure 10.5b). There is a special pathway to take care of the propionyl-CoA,
which is surprisingly complicated (see below).
10.2 Utilization of fatty acids: β-Oxidation 125

a) CH3
+
H3C N CH3
O
CH2
Acyl-carnitine HC O

CH2

COOH

Acyl-CoA CoA S

b) OM IM

Acyl-CoA Acyl-CoA Acyl-CoA

ADP

ATP CAT I CAT II

Fatty acid
CoA Carnitine Carnitine CoA
CoA

Acyl-carnitine Acyl-carnitine

Figure 10.4 Carnitine-mediated transport of fatty acids to the mitochondrial matrix.


a: Structures of acylcarnitine and of acyl-CoA. b: Fatty acids are activated in the cytosol
to acyl-CoA by acyl thiokinase. After transport across the outer mitochondrial mem-
brane, the acyl group is transferred to carnitine by carnitine acyltransferase. Exchange
for free carnitine transports the acylcarnitine molecule into the mitochondrial matrix,
where carnitine is replaced again by coenzyme A by a second acyl transferase.

10.2.3 Reaction mechanisms in β-oxidation

I hope that you noticed the similarities of the enzyme reactions discussed
above to some others we have seen before. At least in the first three steps,
the similarity to reactions in the citric acid cycle—succinate dehydrogenase,
fumarase, and malate dehydrogenase—are quite straightforward. Also notice
the consistent use of redox coenzymes (NAD+ and FAD) in the two pathways:
1. Whereever a CH−OH bond is dehydrogenated, NAD+ or NADP+ is employed
126 10 Triacylglycerol metabolism

a) O β
CoA S ω
α γ FAD
O 1
FADH2
CoA S

O OH 2

CoA S
NAD+
O O 3
NADH++H+
CoA S

CoA-SH
4
O

β
CoA S CH3 O

CoA S

O 1

CoA S

O
b) O

CoA S
CoA S CH3

Acetyl-CoA
O O

CH3
CoA S CoA S C

Propionyl-CoA

Figure 10.5 Reactions in β-oxidation. a: Enzymes: 1, acyl-CoA dehydrogenase; 2,


enoyl-CoA hydratase; 3, hydroxyacyl-CoA dehydrogenase; 4, thiolase. The thiolase reac-
tion yields acetyl-CoA and a new, shortened molecule of fatty acyl-CoA that undergoes
a new round of β-oxidation. b: After completion of β-oxidation, even-numbered acyl
chains will yield acetyl-CoA only, whereas the last unit released from an odd-numbered
acyl chain will be propionyl-CoA.

as the cosubstrate.5 2. Where dehydrogenation occurs across a CH−CH bond,

5 This also holds in other pathways and for CH−NH bonds. The only exception I’m aware of is

the dehydrogenation of glycerolphosphate in the glycerolphosphate shuttle.


10.2 Utilization of fatty acids: β-Oxidation 127

O O

CoA S
+
BH

Enzyme S B
+
H
Enzyme S
O

O
CoA S

CoA S
O
B

Enzyme S

CoA S

Figure 10.6 Mechanism of the thiolase reaction. See text for details.

FAD is employed.
As to the thiolase reaction, we have not seen a completely analogous one.
However, if we look at the individual steps of this reaction, we can still recog-
nize some familiar features (Figure 10.6):
1. The nucleophilic attack of a cysteine sulfur in the active site on a carbonyl
group in the substrate yields a covalent intermediate. This also happens with
glyceraldehyde-3-phosphate dehydrogenase.
2. Acid-base catalysis breaks a carbon-carbon bond adjacent to the carbonyl
bond. Note that the thiolase reaction is reversible. Making of a carbon-carbon
bond adjacent to the carbonyl group by acid-base catalysis then occurs in the
reverse reaction, and we have seen that before with citrate synthase and malate
synthase.
3. The creation of one thioester at the expense of another occurs in the
second step of the pyruvate dehydrogenase reaction.

10.2.4 Utilization of propionyl-CoA

Odd-numbered fatty acids yield one molecule of propionyl-CoA propionyl-


CoAutilization as the final degradation product. This metabolite has an elabo-
rate degradative pathway (Figure 10.7):
1. Initially, propionyl-CoA is converted to S-methylmalonyl-CoA by propi-
onyl-CoA carboxylase. This reaction uses CO2 and ATP; biotin functions as
a coenzyme. The reaction mechanism is the same as in the case of pyruvate
carboxylase (section 7.1).
2. S-methylmalonyl-CoA is converted to its enantiomer R-methylmalonyl-
CoA by methylmalonyl-CoA racemase.
128 10 Triacylglycerol metabolism

Propionyl-CoA Succinyl-CoA
O O

CH3 CH2
CoA S CH2 CoA S CH2 COOH

ATP
CO2
1
3
ADP

O O

H3C 2 H3C
C S CoA C S CoA

COOH COOH

(S)-Methylmalonyl-CoA (R)-Methylmalonyl-CoA

Figure 10.7 Utilization of propinyl-CoA. Enzymes: 1, propionyl-CoA carboxylase; 2,


Methylmalonyl-CoA racemase; 3, Methylmalonyl-CoA mutase.

3. Finally, one carboxyl group is transplanted within the molecule by methyl-


malonyl-CoA mutase to yield succinyl-CoA. This is a frighteningly complex re-
action; we will skip the gory details and just state that it requires Vitamin B12
(cobalamine) as a coenzyme.
Note that succinyl-CoA is a citric acid cycle intermediate. It therefore can
be converted to glucose, so that propionyl-CoA is an exception to the rule that
carbon from fatty acids cannot be used for gluconeogenesis. It seems, however,
that fatty acids with odd numbers of carbons account only for a small fraction
of all fatty acids, so that this exception is not quantitatively very important.

10.2.5 Energy balance of β-oxidation


The energy-rich metabolites accumulated in β-oxidation—NADH, FADH2 , and
acetyl-CoA—are degraded in the TCA and the respiratory chain as described
previously. The exact amount of ATP derived—with the usual assumptions of
3 ATP per NADH and 2 per FADH2 —strikes me as something suitable for an
exam question.

10.3 Triacylglycerol utilization


When triacylglycerol is mobilized from the fat tissue, it is cleaved by an enzyme
named hormone-sensitive lipase. This lipase is stimulated by glucagon and
epinephrin, the antagonist hormones of insulin. The cleavage products, glycerol
and free fatty acids, are released into the plasma, where the fatty acids bind
to albumin for transport. The fatty acids can be utilized in two different ways
(Figure 10.8):
10.3 Triacylglycerol utilization 129

TAG

glycerol
+
FA FA acetyl-CoA

ketone
ketogenesis bodies

acetyl-CoA

gluconeogenesis glucose glycolysis

Figure 10.8 Overview of organ relationships in fat utilization. Triacylglycerol is


cleaved in fat tissue to fatty acids and glycerol. Fatty acids are utilized by β-oxidation
in heart and skeletal muscle, or first converted to ketone bodies in the liver. Ketone
bodies can be consumed by many organs including the brain. Glycerol is utilized for
gluconeogenesis.

1. Directly, that is they are taken up by the energy-requiring target tissue


and degraded by β-oxidation. You will recall that β-oxidation is preceded by
the translocation to the mitochondrion, which requires carnitine. About 95% of
all carnitine is found in skeletal muscle,6 so that it appears that muscle tissue
is the major client for direct utilization. The heart muscle cells also consume
fatty acids by β-oxidation.
2. Indirectly, after initial conversion to ketone bodies in the liver (Figure
10.9). This term comprises two small organic acids, acetoacetate and β-hydrox-
ybutyrate. The brain, which in the well-fed state only utilizes glucose for ATP
regeneration, is able to adapt to ketone bodies during starvation. The brain
is responsible for approximately 20% of our total energy consumption at rest,
and therefore is a major consumer of ketone bodies. Other major consumers
are heart and skeletal muscle.
The second cleavage product of fat, glycerol, is picked up by the liver as
well. It is phosphorylated by glycerol kinase to glycerolphosphate. As in the
glycerolphosphate shuttle (section 6.5), this metabolite is dehydrogenated to
dihydroxyacetone phosphate and can then be utilized for gluconeogenesis.
Glycerol released from fat therefore contributes to keeping up a minimum
supply of glucose in times of starvation.

6 Carnis is Latin for meat – as in chili con carne.


130 10 Triacylglycerol metabolism

10.3.1 Brown fat tissue

You know from experience that fat tissue is mostly white, which is due to
the fact that typical fat cells contain little else than triacylglycerol droplets.
Intensely colored tissues (other than the pigment cells of the skin) are rich
in and owe their color to heme and / or cytochromes. This is the case with
brown fat tissue. The color of this special type of fat tissue derives from
the abundant presence of mitochondria, which in turn contain cytochromes.
They also contain a high amount of an uncoupling protein (section 6.1) called
thermogenin.
In contrast to white fat cells, brown fat cells do not release fatty acids
into the circulation but instead perform β-oxidation themselves. The derived
hydrogen is oxidized in the respiratory chain. However, the resulting proton-
motive force is not used for ATP synthesis but instead simply dissipated as
heat – the purpose of brown fat is heat production. There is little brown fat in
adult humans or most other adult mammals. However, there is a substantial
amount in newborns, who because of their higher surface-to-volume ratio (and
their helplessness) are more prone to hypothermia. Brown fat is also found
in hibernating animal species such as polar bears, which need it to reheat
themselves to operating temperature during arousal from hibernation.

10.4 Ketone bodies


Ketone bodies can be considered a water-soluble and non-toxic transport form
for the carbon mobilized from fatty acids. Ketone body synthesis occurs in the
mitochondria, that is in the same compartment as β-oxidation. It involves the
following reactions (Figure 10.10):
1. Formation of acetoacetyl-CoA from two molecules of acetyl-CoA by thi-
olase (Figure 10.10a). This step is the reversal of the final step in β-
oxidation.
2. Formation of hydroxymethylglutaryl-CoA (HMG-CoA) by HMG-CoA syn-
thase (Figure 10.10b, 1).
3. Release of acetoacetate by HMG-CoA lyase (Figure 10.10b, 2);
4. Reduction of acetoacetate to β-hydroxybutyrate by β-hydroxybutyrate
dehydrogenase (Figure 10.10c).
Acetoacetate can form acetone (CH3 −CO−CH3 ) through slow, spontaneous
decarboxylation. Acetone cannot be metabolized; it is volatile and is exhaled.
A characteristic acetone smell can be detected in persons that utilize fat at a
high rate.
One such situation is diabetic ketoacidosis. If insulin is lacking, fat cells
cannot import glucose. They mistake this for a state of starvation and start
breaking down fat, which is then converted to ketone bodies. If you detect an
10.4 Ketone bodies 131

a) O O O OH

HO HO

b)

TAG

FA + glycerol Acetyl-CoA

Succinate
Acetoacetyl-CoA
TCA
FAD FADH2
Succinyl-CoA Acetoacetate
FA Acetyl-CoA
NADH+H+
NAD+ NADH+H+
NAD+
β-HB β-HB
Acetoacetate

Figure 10.9 Ketone body metabolism. a: Structures of acetoacetate and β- hydroxy-


butyrate (β-HB). b: Overview of metabolic pathways. Triacylglycerol is cleaved to fatty
acids and glycerol in the fat tissue. Fatty acids undergo β-oxidation in the liver. Acetyl-
CoA is converted to acetoacetate, which is released into the blood. Half of the NADH
generated in β-oxidation is used to convert acetoacetate to β- hydroxybutyrate. In the
brain and other tissues, the two substrates are converted back again to acetyl-CoA and
utilized in the citric acid cycle.

acetone smell in an unconscious person, this is highly suspicious of diabetic


coma (see section 14.4).

Another condition with accelerated breakdown of fat is plain ol’ flu and
other states of fever. Metabolism is accelerated by fever; at the same time,
patients often have little or no appetite. Fat is broken down, and again an
acetone smell may develop. This by itself does not indicate a serious condition.

Utilization of ketone bodies in the brain, muscle and other tissues is straight-
forward (Figure 10.9): (1) β-Hydroxybutyrate is dehydrogenated to acetoacetate,
(2) acetoacetate steals coenzyme-A from succinyl-CoA to become acetoacetyl-
CoA, (3) thiolase cleaves acetoacetyl-CoA to acetyl-CoA.
132 10 Triacylglycerol metabolism

a) O
O CoA-SH
+
H
Enzyme S CH3
CoA S CH3
B
Enzyme S
H CH2 S CoA
B
O

O O

H3C S CoA

b) +
Enzyme B H

O O O O

CoA S CH3 CoA S CH3

CH2 S CoA

1
Enzyme-B O

H2O
H CH2 S CoA
1
CoA
O

O OH

O O
2 CoA S CH3

HO CH3 CH2 OH

Acetyl-CoA O

CH3 CH3
c)
NADH+H+ NAD+
C O HO C H

CH2 CH2

C O C O

OH OH

Figure 10.10 Reactions in ketone body synthesis. a: Formation of acetoacetyl-CoA


by thiolase. This reaction is the reversal of the cleavage occurring in β-oxidation.
b: Synthesis of hydroxymethylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase (1) and
release of acetoacetate by HMG-CoA lyase(2). c: Reduction of acetoacetate to D-β-
hydroxybutyrate by β-hydroxybutyrate dehydrogenase.
10.5 Fatty acid synthesis 133

ATP ADP
O O OH

C
CoA S CH3 CoA S C O

CO2

Figure 10.11 The reaction catalyzed by Acetyl-CoA carboxylase.

10.5 Fatty acid synthesis


Fatty acids can be synthesized from acetyl-CoA. This is the major way of uti-
lizing excess dietary carbohydrates. Fatty acid synthesis occurs mainly in the
fat tissue and the liver. Synthesis and degradation of fatty acids are similar in
that they involve a cycle of reactions that changes the length of the substrate
by two carbon atoms at a time. However, they differ from each other in several
aspects:
1. Synthesis runs in the cytosol, whereas β-oxidation occurs in the mito-
chondria. This makes it easier to control the two pathways separately.
2. Synthesis uses NADPH rather than NADH and FADH2 to reduce the C=O
and C=C double bonds. Use of NADPH drives both reactions toward reduction.
3. The C2 subunits that are sequentially added are derived from malonyl-
CoA rather than from acetyl-CoA directly. Again, this thermodynamically fa-
vours synthesis over degradation.
Points 2 and 3 both conspire to make fatty acid synthesis irreversible. In
contrast, β-oxidation is reversible, and reversed β-oxidation is actually used by
mitochondria to synthesize their own fatty acids.
The bulk of the work in fatty acid synthesis is accomplished by fatty acid
synthase, which is quite an amazing molecule: It combines eight distinct cat-
alytic activities on a single polypeptide chain. Its product is palmitic acid
(hexadecanoic acid; sixteen carbons, fully saturated). As stated before, fatty
acids vary in their chain lengths and degree of bond saturation. Chain elonga-
tion and desaturation is accomplished by separate enzymes, called elongases
and desaturases. We just note these here but will not consider them in detail.

10.5.1 Reactions in fatty acid synthesis


The only reaction in fatty acid synthesis that is not carried out by fatty acid
synthase is the first one, which is catalyzed by acetyl-CoA carboxylase. This
reaction is depicted in Figure 10.11. I hope that, by now, you recognize the
pattern: CO2 is fixed, and ATP is expended – another biotin-dependent reaction,
operating in the same way as discussed before for pyruvate carboxylase and
propionyl-CoA carboxylase.
All further reactions are catalyzed by fatty acid synthase. In animals, this is
a large molecule with all active sites on a single polypeptide chain. Two of these
polypeptides form an active dimer. The overall structure shown in Figure 10.12
134 10 Triacylglycerol metabolism

has been determined using electron microscopy and image processing. The
crystal structure has recently been determined as well (Science 321:1315-22,
2008).
The growing fatty acyl chain remains covalently attached to the enzyme
throughout the entire synthesis process. Attachment is through a phospho-
pantetheine group, which provides a flexible tether, enabling the acyl chain to
travel and visit the various active sites on the enzyme in turn (Figure 10.12c).
This is reminiscent of the lipoamide moiety in pyruvate dehydrogenase and
of biotin-dependent carboxylases.7 The phosphopantetheine tether group is
actually not new to us – it also occurs in coenzyme A (Figure 10.12b).
In Figure 10.13, the structure of fatty acid synthase has been rather drasti-
cally simplified; only the two thiol groups, which serve as points of covalent
attachment of acyl-CoA and malonyl-CoA, are shown. Keep in mind, however,
that they are close to each other only in two reactions (3 and 7 in Figure 10.13).
For the other reactions, the tethered acyl residue swings back and forth to in-
teract with the other active sites. The reactions catalyzed by fatty acid synthase
are as follows (see Figure fatFAsynthesis):
1. Transfer of an acetyl-group from acetyl-CoA to the active-site cysteine.
Both in the substrate and the product, the malonyl group is bound as a thioester,
so that this reaction is easy; a similar exchange is part of the thiolase reaction
(Figure 10.10a).
2. Transfer of a malonyl group from malonyl-CoA onto the phosphopan-
tetheine group of the FA synthase. Since the two carriers are so similar, this is
again a very straightforward reaction.
3. Condensation of the malonyl group to the end of the acetyl group. Apart
from the concurrent decarboxylation (which facilitates the transient formation
of a carbanion), this reaction resembles the reversal of the thiolase reaction in
β-oxidation.
4. Reduction of the keto group to a hydroxyl group,
5. Elimination of water to create a C=C double bond and
6. Reduction of the latter to a single bond. These steps are reversals of those
occuring in β-oxidation, except that NADPH is used as the redox coenzyme.
7. Transfer of the extended acyl chain from the phosphopantheteine group
to the aforementioned active site cysteine. Note that this reaction is actually
very similar to the initial transfer of an acetyl group from acetyl-CoA – in both
cases, transfer is from a pantetheine group to the cysteine.
8. The phosphopantetheine site is now free to accept another malonyl
group, and the cycle repeats, with the acetoacetyl group now taking the place
of the acetyl group in the first cycle.
7 In E. coli, the phosphopantetheine group is associated with a separate small protein, the acyl

carrier protein (ACP). In mammalian fatty acyl synthesis, ‘ACP’ is not a separate protein but is
part of the synthase molecule itself.
10.5 Fatty acid synthesis 135

a)

*
*

b) O CH3 H

Enzyme Ser O P O C C C C N C C C N C C S
H2 H H2 H2 H H2 H2
O CH3 OH O O

O O CH3 H

Adenosine P O P O C C C C N C C C N C C S
H2 H H2 H2 H H2 H2
O O CH3 OH O O

c) O

Cys-S-

Figure 10.12 Structure of fatty acid synthase. a: Electron microscopy. Left: The
white squares highlight individual molecules. (No, I can’t recognize them either.) Right:
The three-dimensional structure, obtained by merging and averaging many images of
individual molecules. The two subunits of the dimer are elongated and oriented in
antiparallel fashion. The stars highlight the putative location of the active sites. From:
Brink, Jacob et al. PNAS 99:138-143 (2002). b: Structure of the phosphopantetheine
group (top) compared to coenzyme A. c: The phosphopantetheine group acts as a tether
for the acyl group, which can therefore visit the various active sites of the synthase.
The cysteine is located in one of the active sites (the one which has acyl transferase
activity). Only one of the two enzyme subunits is shown.
136 10 Triacylglycerol metabolism

Pant S

Enzyme Pant S

Cys S Enzyme
1
Cys S CH3
H3C S CoA

O
O
CoA S
Malonyl-CoA

2
CoA S
Malonyl-CoA

Pant S CoA S Pant S O


8
Enzyme Enzyme O O

Cys S CH3 Cys S CH3

O O
7

3 CO2
Pant S CH3

Enzyme O Pant S CH3

Cys S Enzyme O O

Cys S
NADP+
NADPH
6
NADPH 4
NADP+

Pant S CH3 Pant S CH3


5
Enzyme O Enzyme O OH

Cys S H2O Cys S

Figure 10.13 Reactions of fatty acid synthesis. “Pant” represents the pantetheine
moiety of fatty acyl synthase. See text for details.
10.5 Fatty acid synthesis 137

a) b) acetyl-CoA acetyl-CoA
ADP
Mitochondrial Cytosol
5 1 ATP
matrix
citrate citrate
acetyl-CoA acetyl-CoA
ADP oxaloacetate oxaloacetate
1
ATP malate malate 2
2
5 citrate citrate
CoA-SH
NADH+H+ NAD+ NAD+ NADH+H+

OA OA NADH+H+
ADP+Pi
2 NAD+ c) 2 acetyl-CoA 2 acetyl-CoA
4 malate
ATP NADP+ 3 CoA
3 NADPH+H+ acetoacetyl-CoA
1
CO2
2 CoA 2 ADP
CO2 pyruvate pyruvate
CoA
ATP
acetoacetate acetoacetate

Figure 10.14 Mechanisms for the transport of acetyl-CoA from the mitochondrion to
the cytosol for fatty acid synthesis. a: The textbook shuttle. Enzymes: 1, ATP-citrate
lyase; 2, malate dehydrogenase; 3, malic enzyme; 4, pyruvate carboxylase; 5, citrate
synthase. b: A simpler yet more likely shuttle. Enzymes as in a). c: The acetoacetate
shuttle. 1: reactions of ketogenesis; 2: acetoacetyl-CoA synthetase; 3: thiolase.

Finally, when the full length of 16 carbons has been reached, the palmitate
residue is hydrolyzed off the pantetheine residue instead of being transferred
to the cysteine (not shown).
The dimeric nature of the protein raises two possibilities for its mechanism:
(1) The two subunits operate independently, that is the acyl chain only visits
active sites within the subunit to which it is bound. (2) The acyl chain undergoes
reaction in one or more of the active sites on the other subunit.
Splitting the dimer into monomers leaves most of the enzyme activities
intact, indicating that these reactions occur within the same subunit. However,
the initial reaction in each cycle—the condensation reaction between the fully
reduced acyl chain and the next malonyl residue—only occurs in the dimer,
which suggests that it may be catalyzed by the other subunit.

10.5.2 Export of mitochondrial acetyl-CoA


We have noted before that fatty acid synthesis occurs in the cytosol. Since
acetyl-CoA is formed in the mitochondrion by pyruvate dehydrogenase, we face
138 10 Triacylglycerol metabolism

the problem of getting it out of the mitochondrion and into the cytosol.
You may recall that there was a similar problem in β-oxidation. In that case,
the transport of acyl-CoA was accomplished by the carnitine carrier system
(section 10.2.1). Since all reactions in the latter system are reversible and acetyl-
CoA is also an acyl-CoA, we might assume that the carnitine carrier system
could also help us out here. However, if it did transport both acetyl-CoA and
longer acyl-CoAs in both directions, it might set up a futile cycle of fatty acid
synthesis and degradation. Accordingly, acetyl-CoA is transported by other
means. There seem to be several pathways that contribute to this transport
(Figure 10.14):
1. The major mechanism uses the cytoplasmic enzyme ATP-citrate lyase.
Citrate is exported by the tricarboxylate carrier system and cleaved; concomi-
tantly, the acetate fragment is activated to acetyl-CoA using ATP. The problem
then arises how to dispose of the oxaloacetate. While the textbooks usually
account for this as shown in Figure 10.14a, this mechanism is not only com-
plicated but also unbalanced. Citrate can only be transported in exchange
for something. One possibility is malate, which suggests that the mechanism
outlined in Figure 10.14b is more reasonable.
A fairly straightforward transport mechanism that is active at least in mice, but
probably also other mammals, is the export of acetoacetate, which is generated
in the mitochondria by the ketogenesis pathway (Figure 10.10). Acetoacetate
can be converted back to acetyl-CoA by cytosolic acetoacetyl-CoA synthetase,
which uses ATP, and a thiolase (Figure 10.14c).
The acetoacetate shuttle has the advantage of the lowest ATP expenditure
(one ATP per two molecules of acetyl-CoA translocated).

10.5.3 Supply of NADPH for fatty acid synthesis


While the hexose monophosphate shunt typically gets the most credit for
NADPH production, it seems that fat cells and possibly also liver cells ob-
tain most of their NADPH by an alternative mechanism that uses cytoplasmic
NADP+ -dependent isocitrate dehydrogenase (Figure 10.15). Isocitrate formed
in the TCA can be translocated into the cytosol in exchange for α-ketoglutarate.
The isocitrate dehydrogenase reaction produces NADPH in the cytosol and also
replaces the α-ketoglutarate. The mitochondrial isocitrate dehydrogenase step
is simply skipped, and the TCA resumes at the level of α-ketoglutarate.
Now, Watson, if you have been following closely, you will have observed that
this will only satisfy half the need for NADPH, since we get only one mole of
isocitrate per mole of acetyl-CoA, but fatty acid synthesis requires two moles
of NADPH per acetyl-CoA.
NADH/NADP+-transhydrogenase to the rescue: In Figure 6.14, it was de-
scribed how transhydrogenase and NADP+ -dependent isocitrate dehydrogenase
can conspire to regenerate isocitrate from α-ketoglutarate. Instead of being
10.6 Pharmacological inhibition of fatty acid synthase 139

Mitochondrial matrix Cytosol

citrate

NADP+ isocitrate isocitrate


NADP+

NADPH+H+
NADPH+H+ α-ketoglutarate α-ketoglutarate + CO2

succinyl-CoA

Figure 10.15 The role of cytosolic isocitrate dehydrogenase in the regeneration of


NADPH for fatty acid synthesis. See text for details.

consumed in a futile cycle as discussed then, this isocitrate can again be made
available for cytosolic NADPH regeneration.

10.6 Pharmacological inhibition of fatty acid synthase


Cerulenin is a fungal antibiotic that binds to and irreversibly inhibits fatty acid
synthase. Its structure resembles the β-keto-acyl intermediates (Figure 10.16a),
and the active site to which cerulenin binds is the very same one that forms
the β-keto-acyl group by condensation. The cysteine in this site then reacts
with the epoxide ring in the cerulenin and is alkylated. While epoxide drugs
are not terribly popular because they are quite reactive and often toxic, there
is presently some interest in the development of fatty acid synthase inhibitors,
modeled on the structure of cerulenin, for the treatment of obesity. While this
does not strike me as a compelling idea,8 a more interesting application is in
the treatment of cancer. Most non-cancerous cells do not express fatty acid
synthase, relying instead on fatty acids supplied by the liver and fat tissue.
However, fatty acid synthase expression has been observed in cancer cells; the
fatty acids they produce probably feed their synthesis of membrane glyco- and
phospholipids, which they require for proliferation. Inhibition of fatty acid
synthase can induce apoptosis (programmed cell death) in tumor cells.
Figure 10.16b shows an experimental synthetic drug that is somewhat sim-
ilar to polyphenols occurring in green tea and other natural sources. In mice
8 I look forward to the discoveries of metabolic derailments these may cause in the face of

continued excess calory intake – what is going to become of the surplus acetyl-CoA if fatty acid
synthesis is inhibited: Ketone bodies? Cholesterol? Will glycolysis be backed up and diabetes be
induced? All of the above?
140 10 Triacylglycerol metabolism

a)
OH

O O

+
H

O S Cys Enzyme

NH2

O O

OH S Cys Enzyme

NH2

O O

b)
avg. tumor volume (mm3)

OH
500
HO OH
400 untreated

300
O O
200
O 100 compound 7
OH
O 0
OH 0 10 20 30 40
compound 7 OH days of treatment

Figure 10.16 Inhibitors of fatty acid synthase. a: Cerulenine. Top: Structure of a


β-keto-fatty acid (shown for comparison). Middle: Reaction of the active-site cysteine
of fatty acid synthase with the epoxide moiety of cerulenin. Bottom: The resulting
inactivated state. b: Structure of and tumor growth inhibition by an experimental fatty
acid synthase inhibitor. The plot shows the growth of tumor grafts in mice, with and
without treatment with the inhibitor. Redrawn from original data in Puig et al., Clin.
Cancer Res. 15:7608-7615 (2009).

experiments, it substantially reduces the growth rate of transplanted cancer.


While such experimental cancers are notorious for being easier to treat than
actual clinical cancer in humans, the results suggest that this line of research
is worth pursuing.
Chapter 11

Cholesterol metabolism

Contrary to popular belief, the biological role of cholesterol is not limited to


being the bad guy. Instead, it has a number of essential physiological functions:
1. In each individual cell, it occurs as a major constituent of the plasma
membrane. Cholesterol controls physical properties of the membrane, which
are important for the function of membrane proteins such as receptors and
transporters. Depletion of membrane cholesterol cripples many functions of
cells.
2. Cholesterol is the precursor of bile acids, which are essential for fat
digestion.
3. Cholesterol is the precursor of all steroid hormones: Androgens, estro-
gens, gestagens, glucocorticosteroids, mineralocorticoids, and calciferols. Some
examples are illustrated in Figure 11.1.
Cholesterol can both be obtained from the diet and be synthesized in human
metabolism. However, we cannot degrade it; therefore, cholesterol has no
significant role in energy metabolism. I’m going to cover it here anyway because
its relationship to fat metabolism and its medical and physiological relevance.

141
142 11 Cholesterol metabolism

Table 11.1 Characteristics of the major lipoproteins. VLDL: Very low density lipopro-
tein; LDL and HDL: Low and high density lipoproteins.

Chylomicrons VLDL LDL HDL


Density (g/ml) 0.95 0.95–1.0 1.02–1.06 1.06–1.12
Origin Small intestine Liver VLDL Liver
Role in Transport of Distribution of Distribution Transport of
transport dietary fat triacylglycerol of cholesterol excess
from liver from liver cholesterol to
liver
Most Triacylglycerol Triacylglycerol Cholesterol Protein,
abundant phospholipids,
constituents cholesterol

11.1 Uptake and transport of cholesterol


The structure of cholesterol (Figure 11.1, top left) has very little similarity with
triacylglycerol. However, they are both hydrophobic molecules, and therefore
the two lipids share the same major mechanism of transport in the bloodstream,
which is by way of lipoproteins. This applies to both dietary and endogenously
synthesized lipids.
Cholesterol that is taken up from the intestines initially becomes bound to
chylomicrons. It may then be transferred from there to cells in the periphery—
recall that chylomicrons bypass the liver—or be redistributed to other lipopro-
tein species. Some cholesterol will remain with the chylomicron remnants that
are finally phagocytosed and degraded in the liver.

11.2 Distribution of cholesterol from the liver to the pe-


riphery
The major site of cholesterol biosynthesis is—once more—the liver.1 The ma-
jor lipoprotein species synthesized by the liver is known as very low density
lipoprotein (VLDL), which transports both cholesterol and triacylglycerol from
the liver to the periphery. By extraction and exchange of triacylglycerol, the
composition of circulating VLDL successively changes until it becomes low den-
sity lipoprotein (LDL), which has a particularly high concentration of cholesterol.
This lipoprotein species is removed from the circulation through endocytosis2
by cholesterol-requiring cells, for example in tissues that synthesize steroid
hormones. Endocytosis requires a specific receptor, the LDL receptor. A genetic
1 The liver has been dubbed the lab of the human body. I disagree: It is more productive, and

it makes fewer mistakes than any lab I’ve seen so far.


2 Note that uptake by cells other than vascular endothelia must be preceded by transcytosis—

endocytosis followed by exocytosis on the opposite side—across the endothelial cells.


11.3 Transport of excess cholesterol to the liver 143

CH2OH
H3C CH3
C O
CH3 HO CH3
CH3 OH

CH3 CH3

H H H H

HO O

Cholesterol Cortisol

O CH3
C OH
CH3 CH3

CH3

H H H H

O HO

Progesterone Estradiol
O

O
OH
OH
CH3
CH3

CH3
CH3

H H

O
HO O

Testosterone Cholic acid


Figure 11.1 Structures of cholesterol and of several molecules derived from it. Cor-
tisol, a glucocorticoid, induces enzymes that increase blood glucose, such as those
necessary for gluconeogenesis. Progesterone, estradiol, and testosterone are sexual
hormones. All these hormones are steroid hormones. Cholic acid is a detergent and is
important in intestinal fat solubilization.

deficiency of this receptor is associated with increased blood levels of LDL and
increased risk of heart attack and stroke (see section 11.9).

11.3 Transport of excess cholesterol to the liver

Excess cholesterol can be transported back from peripheral tissue to the liver
by high density lipoprotein (HDL). Among all lipoproteins, this one has the
highest ratio of protein to lipid and therefore the highest density. A high
level of HDL in the blood is associated with a decreased risk of cardiovascular
disease; therefore, HDL has been dubbed the ‘good cholesterol’ as opposed to
LDL, the ‘bad cholesterol’. The liver can either recycle the cholesterol into new
144 11 Cholesterol metabolism

lipoproteins, or dispose of excess cholesterol through secretion into the bile


(see later).

11.4 Esterification of cholesterol


Unmodified cholesterol has a preference for a superficial location in lipid
phases (e.g., membranes), such that its polar hydroxyl group peeks out into
the aqueous phase. This −OH group can be esterified, so that it becomes
shielded by a fatty acyl residue (Figure 11.2). The fatty acyl residue is pur-
loined from phosphatidylcholine, one of the major phospholipids in mem-
branes and lipoproteins. The enzyme catalyzing this exchange is known as
lecithin:cholesterol acyltransferase (LCAT). Cholesterol esters partition into the
interior of lipoprotein particles, which greatly increases the particles’ transport
capacity for cholesterol.

11.5 Synthesis of cholesterol


Cholesterol biosynthesis occurs in animals but not in plants or fungi. The latter
have similar sterols for similar purposes, which however cannot be converted to
cholesterol. Therefore, it is essential for animals—particularly for plant-feeding
ones, such as sheep, goat and vegetarians—to have a pathway for cholesterol
biosynthesis. This pathway is quite complex, involving some 30 enzymes, and I
don’t know them all myself. We will not look at all reactions in the biosynthesis
individually but only at the initial stages, up to lanosterol, which is the first
sterol intermediate.
The first steps of the synthesis tie in with other metabolic pathways we
have seen before (Figure 11.3). Synthesis starts with Acetyl-CoA in the mito-
chondrion, which is used to synthesize hydroxymethylglutaryl-CoA (HMG-CoA).
These reactions also occur in ketogenesis (section 10.4). However, while the
entire process of ketogenesis occurs in the mitochondrion, the formation of
HMG-CoA in sterol synthesis occurs in the cytosol; therefore, we have separate
versions of HMG-CoA synthase in the two compartments.
All subsequent steps occur in the smooth endoplasmic reticulum. HMG-
CoA reductase reduces HMG-CoA to mevalonate, which in turn is converted to
various isoprene compounds. Several rounds of polymerization3 lead to the
linear hydrocarbon molecule squalene, which is then converted to lanosterol. A
lengthy series of subsequent modifications leads to cholesterol.
The reactions of the synthetic pathway are shown in Figure 11.4. The reac-
tion catalyzed by HMG-CoA reductase is the first committed step, which means
3 I’m using the term loosely – the reaction mechanisms are different from typical chemical

polymerization.
11.5 Synthesis of cholesterol 145

a) CH3
+ H3C CH3
H3C N CH3
CH3
CH3

O H CH3

H H

O O O

O O O

LCAT

b) free cholesterol

O O

O
cholesterol ester
O

Figure 11.2 Esterification of cholesterol. a: One acyl chain of phosphatidylcholine


(left) is transferred to cholesterol by LCAT (lecithin:cholesterol acyltransferase). b:
Distribution of free cholesterol and of cholesterol esters in lipoprotein particles. The
free cholesterol is confined to the surface; the esters distribute to the interior, which
increases the transport capacity.

that from this point onwards the substrates have no other option than becom-
ing a sterol.4 Therefore, HMG-CoA reductase is the main target of regulatory
4 No quantitatively important one, at least. However, farnesyl-pyrophosphate is also used in
146 11 Cholesterol metabolism

HMG-CoA-
reductase
Acetyl-CoA HMG-CoA Mevalonate (C6)

3 ATP

3 ADP CO2

Activated C15 Activated C10 Activated C5

Squalene (linear C30) Lanosterol (C30) Cholesterol

Figure 11.3 Overview of cholesterol biosynthesis. The first committed step is the
reduction of HMG-CoA to mevalonate, which then is activated by ATP for polymeriza-
tion up to squalene. Squalene, the last linear metabolite, is converted to lanosterol, the
Mother of All Sterols.

mechanisms, which in turn is exploited in pharmacotherapy (see later).


The product of HMG-CoA reductase, mevalonate, undergoes repeated phos-
phorylations. One of the phosphate groups is used together with decarboxyla-
tion to introduce a C=C double bond; the other ones are used later in polymer-
ization. The most interesting reaction is the cyclization of squalene, the final
polymerization product. Following the introduction of an epoxy group, all but
one of the double bonds are rearranged, resulting in the formation of the first
sterol, lanosterol. The enzyme in question, squalene monooxygenase, belongs
to the cytochrome P450 family.
Several more steps are required to convert lanosterol to cholesterol. The
last intermediate, that is the immediate precursor of cholesterol, is 7-dehydro-
cholesterol, which is also the precursor of cholecalciferol (see below).

11.6 The endoplasmic reticulum in steroid synthesis


As noted above, the site of all reactions in cholesterol biosynthesis except the
first one is the smooth endoplasmic reticulum. This also applies to most fur-
ther modifications that convert cholesterol to one or the other steroid hormone.
The smooth ER hosts many different cytochrome P450 enzymes. One of these
is squalene monooxygenase; others are responsible for introducing the vari-
ous hydroxyl and carbonyl groups into the sterol molecule in the synthetic
pathways for the steroid hormones (Figure 11.1).
Many intermediates in the synthesis of cholesterol and of its derivatives
are very hydrophobic and very poorly soluble in water; they therefore have to
reside in an apolar environment. On the other hand, the enzymes, like almost
the post-translational modification of some membrane-associated proteins.
11.6 The endoplasmic reticulum in steroid synthesis 147

O CH3 O CoA-SH O CH3 3 ATP 3 ADP O CH3


CoA S C C C C O O C C C C OH O C C C C O P P
H2 H2 H2 H2 H2 H2 H2 H 2
OH OH O
1 2 P
HMG-CoA 2 NADPH+H+ 2 NADP+ Mevalonate 3-P-5-PP-mevalonate

3
CO2 P

Dimethylallyl-PP CH3 Isopentenyl-PP CH3


H3C C C O P P H2C C C O P P
H H2 4 H2 H2

5
P-P

Geranyl-PP CH3 CH3


H3C C C C C C O P P
H H2 H2 H H2
6

CH3 CH3 CH3


Farnesyl-PP H3C C C C C C C C C O P P
H H2 H2 H H2 H2 H H2

NADPH+H+

7
P-P P-P NADP+

NADPH+H+ O2 squalene

8
NADP+ H2O

+
C

HO HO lanosterol
O
+
H
9

Figure 11.4 Enzyme reactions in the synthesis of lanosterol, the sterol precursor
of cholesterol. PP stands for pyrophosphate. Enzymes/reactions: 1, HMG-CoA reduc-
tase; 2: Mevalonate kinase and phosphomevalonate kinase; 3: Phosphomevalonate-5-
pyrophosphate decarboxylase; 4: Isopentenyl-pyrophosphate isomerase; 5: Geranyl-
pyrophosphate synthase; 6: Farnesyl-pyrophosphate synthase; 7: Squalene synthase; 8:
Squalene monooxygenase. 9: Reaction intermediates of the squalene monooxygenase
reaction.
148 11 Cholesterol metabolism

Mitochondria

Smooth endoplasmic reticulum

Rough endoplasmic reticulum

Figure 11.5 Electron microscopy of the endoplasmic reticulum in steroid-hormone


synthesising cell. The organelle is delineated by highly convoluted membranes,
wherein the reactions of sterol modifications proceed. Similarly, in many reac-
tions in cholesterol synthesis, the hydrophobic substrates are located within the
apolar membrane core. Reproduced, with permission from Roger Wagner, from
http://www.udel.edu/Biology/Wags/histopage/histopage.htm.

all proteins, are at least partially polar and cannot immerse completely inside
the same apolar environment. The solution to this problem is to perform the
reactions at the interface of polar and apolar environments, that is at membrane
surfaces. This means that the membrane of the smooth ER is not just there to
delimit a separate compartment, but itself is a reaction compartment.
The membrane of the smooth endoplasmic reticulum is extensively bulged
and invaginated, which gives it a very large surface area. The throughput of
cholesterol synthesis should be proportional to this surface area. Accordingly,
the development of the smooth ER is particularly prominent in cells that per-
form sterol or steroid hormone synthesis (Figure 11.5).

11.7 7-Dehydrocholesterol and vitamin D3


An interesting branch of the cholesterol synthesis pathway leads to the synthe-
sis of vitamin D3 (calciferol). Since it vitamin D3 can by synthesized in human
metabolism, it is not strictly a vitamin. The compound is synthesized from
7-dehydrocholesterol, which is the immediate precursor of cholesterol. The
conversion is a photochemical reaction: It requires the absorption of a UV pho-
ton and therefore can only occur at the body surface, that is in the skin. Two
11.8 Regulation of cholesterol synthesis 149

successive enzymatic hydroxylations, which do not require any more UV light,


then convert calciferol to 1,25-dihydroxycalciferol. This molecule is essential
in the uptake of calcium and phosphate from the gut.
A lack of 1,25-dihydroxycalciferol causes ricketts, a disease characterized by
bone deformations.5 The importance of 1,25-dihydroxycalciferol is responsible
for the variation of human skin colors. While dark pigment protects the skin
from damage by UV irradiation, the UV photons gobbled up by the pigment are
not available for the synthesis of calciferol. When Homo sapiens left Africa for
less sunny climates, the shortage of sunshine created a selective pressure for
lighter skin, which increases the availability of photons for calciferol synthesis.
With the availability of calciferol as a food supplement, light skin color does
no longer seem to have any biological advantages – only the disadvantage of
higher rates of skin cancer. However, it is probably a good idea for dark-skinned
people living in northern climates to be particularly conscientious about giving
calciferol (vitamin D) prophylaxis to their children.

11.8 Regulation of cholesterol synthesis

The biosynthesis of cholesterol, like that of many other metabolites, is regu-


lated both by allosteric control of enzyme activity and by enzyme induction.
The committed step is the reduction of HMG-CoA to mevalonate, and therefore
it makes sense that HMG-CoA reductase is the primary target of regulation. The
enzyme is allosterically inhibited by the final product, that is by cholesterol
itself.
The genetic regulation of cholesterol synthesis has been worked out fairly
recently, and it operates by quite a neat mechanism. Several membrane proteins
participate in it (Figure 11.6a): The Sterol Response Element Binding Protein
(SREBP), the SREBP Cleavage Activating Protein (SCAP), and two SREBP-specific
proteases (S1P and S2P). SCAP is the actual cholesterol sensor that changes
conformation in response to varying cholesterol concentrations within the ER
membrane. If the level of cholesterol is high, SCAP does not bind SREBP, and
SREBP is left behind when SCAP relocates from the ER to the Golgi apparatus
(Figure 11.6b). Nothing further happens in this case. The action starts when
the concentration of cholesterol in the ER membrane is low (Figure 11.6c):
1. SCAP adopts a different conformation that now binds SREBP, dragging it
along for the ride to the Golgi apparatus.
2. In the Golgi, S1P and S2P lie in ambush fo SREBP and cleave it.
3. The major SREBP fragment is now free to leave the membrane and to
translocate to the nucleus.
5 . . . and, if we are to believe current news, cancer, depression, earthquakes, and about 98% of

all other evil befalling mankind.


150 11 Cholesterol metabolism

a) S1P SREBP SCAP

b)

ER

Golgi

c)

ER

Golgi

NM

SRE
+

RNA polymerase

HMG-CoA reductase mRNA

Figure 11.6 Transcriptional regulation of cholesterol synthesis. a: The elements


that participate in it. SRE: Sterol response element; SREBP: SRE binding protein; S1P:
SREBP-specific protease 1; SCAP: SREBP cleavage activating protein. b: Situation at high
cholesterol concentrations. c: The processes occurring at low cholesterol concentration.
See text for details.
11.9 Cholesterol in atherosclerosis 151

HO HO
COOH COOH

OH OH
O

Figure 11.7 Structure of lovastatin, an inhibitor of HMG-CoA reductase (left) and of


mevalonate (right).

4. In the nucleus, SREBP binds to sterol response elements (SREs). These are
short specific DNA sequences that occur in various places of the genome.
5. One SRE is located upstream of the gene of HMG-CoA reductase. Binding
of SREBP to this element increases transcription and therefore the overall
activity of HMG-CoA reductase.
Induction of HMG-CoA reductase will increase the rate of cholesteorl synthe-
sis, which will ultimately suppress the further proteolytic activation of SREBP.

11.9 Cholesterol in atherosclerosis


The level of plasma cholesterol, and in particular LDL-associated cholesterol, is
one of the main risk factors of atherosclerosis, the degenerative vascular disease
underlying myocardial infarction and stroke. In western countries, this is the
leading cause of death, being more common than all cancers and leukemias
combined. Briefly, atherosclerotic lesions develop as follows:6
1. Small mechanical lesions in the vascular endothelium—the innermost
layer of blood vessel walls—allow leakage of blood plasma into the muscular
layers beneath. Formation of these small leakages is thought to be promoted
by high blood pressure.
2. The plasma lipoproteins that leaked into the tissue are degraded. Because
of its low solubility, cholesterol that is released from degraded lipoproteins
precipitates.
3. The cholesterol particles trigger invasion of phagocytic cells and in this
way contribute to triggering inflammation, which in turn increases the tissue
damage and turns the small, potentially reparable defects of the vessel wall
into large lesions.
A crucial feature of the atherosclerotic lesion is the erosion of the endothe-
lium, which normally inhibits blood coagulation. Stroke or infarction occurs
when blood coagulates at the site of such a lesion. Coagulated blood clogs the
artery and suffocates the tissue downstream.
6 There are other contributing factors, possibly including infectious agents.
152 11 Cholesterol metabolism

The main factors that may induce a high plasma cholesterol concentration
are (1) the diet: Rich in cholesterol, or in fat or excess carbohydrates, which may
be converted first to acetyl-CoA and then to cholesterol, (2) physical inactivity:
Sedentary lifestyles, such as typical of prison or university inmates, (3) genetic
factors, in particular a defect of the LDL receptor.
The latter condition is particularly serious if it is homozygous, that is if the
receptor is completely absent. The levels of LDL and cholesterol in the plasma
can reach several times the normal level. The rate of progress of atherosclerosis
and the risk of stroke and myocardial infarction are greatly increased, so much
so that the typical life expectancy of homozygous patients is only 30-40 years.
Heterozygous patients still are at significantly higher risk than the average
person, but the condition is more tractable with the available means of therapy.

11.10 Therapy of hypercholesterolemia


The obvious way of treating the various forms of hypercholesterolemia is to try
and lower the blood cholesterol level. What therapeutic principles are available
to do this? Several:
1. Limiting the dietary intake of cholesterol. Since cholesterol occurs only
in animals but not plants, an obvious way of restricting intake of cholesterol is
or reduction of meat and eggs in favour of fruit and vegetables.
2. Vegetables are also rich in poly-unsaturated fatty acids (those with sev-
eral C=C double bonds) and in carbohydrate fibers, which seem to have a
greater favourable effect than restriction of cholesterol itself. The mechanism
by which these beneficial effects arise are not well understood.
3. Inhibiting the intestinal uptake of cholesterol. This can be done with
sitosterol, a plant sterol that has no role in human metabolism. The mecha-
nistic aspects of this inhibition are not clear but it appears to be some kind of
competition, which in turn suggests a specific uptake mechanism for choles-
terol. Uptake can also be inhibited by ezetimibe and other recently developed
drugs. The target protein in the intestinal membrane that ezetimibe binds to
has been identified. It is known as the Niemann-Pick C1-like protein 1 (NPC1L1).
Ezetimibe interferes with the endocytosis of this protein; it may be that uptake
of cholesterol depends on this same endocytotic process.
4. Inhibition of endogenous cholesterol synthesis. The most important class
of synthesis inhibitors are the statins, for example lovastatin, which mimic
mevalonate in structure and block the active site of HMG-CoA reductase (Figure
11.7).
5. Promotion of cholesterol elimination, by way of bile acid depletion. As
stated above, bile acids are derived from cholesterol. Out of the consider-
able amount of bile acids secreted—several grams per day—approximately
95% are taken up again in the terminal ileum (the lowermost section of the
small intestine) and recycled. Cholestyramine is a granulated polymer that
11.10 Therapy of hypercholesterolemia 153

exposes hydrophobic and cationic binding sites, which combine avidly with the
hydrophobic and anionic moieties of bile acid molecules. Ingested cholestyra-
mine will bind bile acids and simply take them along down the pipe. To replace
the lost bile acids, the liver will increase their synthesis and therefore deplete
the pool of cholesterol.
Apart from bile acids, excess cholesterol itself is also secreted into the bile,
where it depends on bile acids to remain in solution. A drawback of cholestyra-
mine therapy is that, by depleting bile acids, it promotes the precipitation of
cholesterol, favouring the formation of cholesterol bile stones.
While for maximum effect the different principles are often combined, inhi-
bition of synthesis with statin drugs is the most effective single principle and
currently has a prominent place in therapy.
Chapter 12

Amino acid metabolism

We have seen before that during digestion in the intestines proteins are bro-
ken down to their constituent amino acids. Proteins contain twenty standard
amino acids, which are incorporated into them during translation, and several
non-standard ones that are mostly formed by post-translational modification.
These are much less abundant than the standard amino acids and will not be
considered here.
Amino acids, in human metabolism, have three main usages:
1. As building blocks for our own protein synthesis. Animals, including
humans, are essentially parasites and have a lazy synthetic metabolism. Ac-
cordingly, we possess synthetic pathways for only 10 out of the 20 standard
amino acids. The residual 10 amino acids have to be obtained from the diet
and are called the essential amino acids.
2. As a source of energy. Depending on the composition of the diet, this
role may be very significant. Carbohydrates occur nearly exclusively in plant-
derived foodstuffs. Therefore, on a meat-only diet, amino acids become the
major source of glucose.
3. As building blocks for other things such as nucleotides and heme.
We will here focus energy metabolism, that is on the degradation of amino
acids. Synthesis will be skipped, except for some simple examples that involve
no more than transamination. We only note that nine out of twenty amino acids
cannot be synthesized in mammalian cells and therefore have to be obtained
with the diet. These essential amino acids are histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, threonine, tryptophane, and valine. Arginine
is synthesized but apparently not in sufficient amounts and often listed as a
tenth essential amino acid.

154
12.1 Overview of amino acid degradation 155

Arg, Gln, Glu, His, Pro α-Ketoglutarate

Ile, Met, Thr, Val Propionyl-CoA Succinyl-CoA

Phe, Tyr Fumarate

Asn, Asp Oxaloacetate Glucose

Ala, Cys, Gly , Ser Pyruvate

Leu, Lys, Phe, Trp, Tyr Acetoacetate Acetyl-CoA

Figure 12.1 Overview of amino acid degradation. Glucogenic amino acids that give
rise to either pyruvate or TCA intermediates that can be turned into glucose. Ketogenic
amino acids give rise to acetoacetate and acetyl-CoA, which do not yield glucose.

12.1 Overview of amino acid degradation

All amino acidsdegradation amino acids contain at least one nitrogen atom,
which forms their α-amino group.Nitrogen has no use in energy metabolism
and has to be eliminated. There are two key processes in metabolic nitrogen
elimination: 1. Transamination removes the α-amino group from one amino
acid and transfers it to α-ketoglutarate. This leads to the accumulation of
glutamate. 2. The release of nitrogen from glutamate and its conversion to
urea. This is accomplished by the urea cycle in the liver.
Several amino acids—arginine, asparagine, glutamine, histidine, lysine, pro-
line, tryptophan—contain additional nitrogen atoms in their side chains. For
these, adapter pathways exist that ultimately also feed the nitrogen into the
urea cycle.
Removal of nitrogen is typically an early step in amino acid degradation and
leaves behind the carbon skeleton. The structure of the latter is different for
each amino acid, and accordingly each amino acid has its specific pathway of
degradation. However, they can be grouped into two broad classes. As Figure
12.1 shows, most amino acids can be converted to intermediates of the citric
acid cycle or to pyruvate. These are called glucogenic amino acids, since they
can contribute to the synthesis of glucose (gluconeogenesis). Those that yield
acetoacetate are called ketogenic, since acetoacetate is one of the two ketone
bodies (see section 10.4).
What happens to glucogenic amino acids when they are available in excess
over the demand for glucose? This may well happen if we are on a very protein-
rich diet. One option would be to first convert all substrates to oxaloacetate
156 12 Amino acid metabolism

in the citric acid cycle and then short-circuit gluconeogenesis and glycolysis at
the level of phosphoenolpyruvate:

Oxaloacetate + GTP ------→


- PEP + GDP + CO2
PEP + ADP ------→
- pyruvate + ATP

Although this would seem reasonably straightforward, this pathway is ap-


parently not quantitatively important. Instead, the substrates are drained from
the TCA cycle at the level of malate by malic enzyme:

Malate + NADP+ ------→


- pyruvate + NADPH + H+ + CO2

An advantage of this pathway is that it yields NADPH and not NADH, as is


the case in the formation of oxaloacetate by malate dehydrogenase. Excess
pyruvate is mostly converted to acetyl-CoA by pyruvate dehydrogenase and
used for fatty acid synthesis; malic enzyme provides part of the NADPH needed
by fatty acid synthase.

12.2 Transamination
In the degradation of most standard amino acids, an early step in degrada-
tion consists in transamination, which is the transfer of the α- amino group
from the amino acid to an α-keto acid. There are several different aminotrans-
ferases, each of which is specific for an individual amino acid or for a group
of chemically similar ones, such as for example the group of branched amino
acids, which includes leucine, isoleucine, and valine. With all these enzymes,
the α-keto acid that accepts the amino group is always α-ketoglutarate (Fig-
ure 12.2). Transamination is freely reversible; therefore, both glutamate and
α-ketoglutarate are substrates of every single transaminase. If amino groups
are to be transferred between two amino acids other than glutamate, this will
still occur by transient formation of glutamate (Figure 12.2b).
The mechanism of transamination is depicted in Figure 12.4 for alanine, yet
is the same with all transaminases. The reaction occurs in two stages:
1. Transfer of the amino group from alanine to the enzyme, which releases
pyruvate, and
2. Transfer of the amino group from the enzyme to α-ketoglutarate, which
releases glutamate.
In Figure 12.4, only the first half-reaction is shown. The second half-reaction is
the exact reversal of the first one; this also implies that the entire reaction is
reversible. Overall, the mechanism consists in the first substrate arriving and
leaving before the second substrate enters and leaves; this is dubbed a ping
12.2 Transamination 157

a) COOH COOH COOH COOH

C O H2N CH H2N CH C O

CH2
+ CH3 CH2
+ CH3

CH2 CH2

COOH COOH

b) COOH COOH COOH

H2N CH C O H2N CH

CH3 CH2 CH2

CH2 COOH

COOH

1st transaminase 2nd transaminase


COOH

H 2N CH COOH
COOH CH2 C O
C O CH2 CH2
CH3 COOH COOH

Figure 12.2 Transamination reactions. a: Glutamate pyruvate transaminase (also


called alanine amino transferase) transfers the α-amino group from alanine to α-
ketoglutarate, which yields glutamate and pyruvate. b: All transaminases have α-
ketoglutarate as one of their substrates. Transfer of amino groups between arbitrary
amino and α-keto acids (here: alanine and oxaloacetate) occurs by transient transfer to
α-ketoglutarate.

pong bi bi reaction (Figure 12.3). 1 While two different substrates must be used
for the reaction to have a net effect, it is of course possible for amino acid 1
and amino acid 2 to be identical – the reaction will work just fine, but simply
achieve no net turnover.
The reaction mechanism revolves around the coenzyme pyridoxal phos-
phate (PLP):
1. At the outset of the reaction, PLP is bound as a Schiff base to the -amino
group of a lysine residue in the active site (Figure 12.4a).
2. The bond between PLP and the enzyme is separated, and PLP forms a
Schiff base with the amino acid substrate instead (Figure 12.4b, steps 1 and 2).
3. The liberated lysine residue abstracts the α hydrogen as a proton (step
3), and the electron left behind travels all the way down to the nitrogen at the
bottom of the PLP ring. PLP is often said to act as an electron sink. This has
the effect of turning the bond between the α carbon and the α nitrogen into a
Schiff base.

1 The mind boggles when one tries to figure out what kind of greek nomenclature Old World

biochemists could have dreamed up for this behaviour. Maybe something like: Amphiballistic
antidromic sequential?
158 12 Amino acid metabolism

amino acid 1 α-keto acid 1 α-keto acid 2 amino acid 2

E-Lys-N=PLP E-Lys-NH2 E-Lys-N=PLP


pyridoxamine

Figure 12.3 The ping pong bi bi kinetic mechanism of transamination. The two
substrates arrive and leave one after another; the second half-reaction (dotted blue
arrow) is the exact reversal of the first one (solid blue arrow), save that a different
α-keto is used.

4. The Schiff base is hydrolyzed to yield the α-keto acid and the amino
derivative of PLP, called pyridoxamine phosphate (steps 4 and 5).
The PLP in its various forms stays within the active site throughout, even
while not bound to the enzyme covalently. As stated above, the second half
reaction is the exact reversal of the first, and a good exercise for you would be
to draw the individual steps yourself.

12.3 The urea cycle


While transamination solves the problem of removing the α-nitrogen for the
amino acids other than glutamate, there also must be a mechanism to regener-
ate the α-ketoglutarate that is converted to glutamate in each transamination
reaction. This is accomplished in the glutamate dehydrogenase reaction, which
releases the nitrogen as ammonia (Figure 12.7c).
Ammonia is quite toxic, and its levels in the systemic circulation are kept in
the micromolar range.2 The glutamate dehydrogenase reaction runs primarily
in the liver. There, the ammonia is scooped up immediately by the urea cycle,
which converts it to the much less toxic metabolite urea. Urea is released into
the circulation and then eliminated through the kidneys. The overall reaction
of the urea cycle is:

NH3 + HCO− - (NH2 )2 CO + fumarate


3 + Aspartate ------→

with the simultaneous expenditure of several molecules of ATP in order to


make this happen.

12.3.1 Reactions in the urea cycle


The urea cycle involves the following reactions (Figure 12.5):
2 In liver cirrhosis, one of the main problems is the lacking capability of the liver to detoxify

ammonia derived from bacterial metabolism in the large intestine. Antibiotics are used in this
condition to reduce bacterial growth and ammonia formation.
12.3 The urea cycle 159

a) Figure 12.4 Mechanism


Enzyme of transamination. a: Struc-
CH2 ture of pyridoxalphosphate
+ (PLP), free and covalently
O NH
HC HC bound to the enzyme. b:
H2 H2 Details of the mechanism,
C O C O
P P shown for the first half
reaction only. At the end
HC HC
N
+
CH3 N
+
CH3 of this reaction, PLP retains
H H
the former α-nitrogen, and
the substrate is released
b) as an α-keto acid (Figure
12.3). The second half of
H Enzyme H Enzyme the reaction is the exact
H3C C COOH H3C C COOH
CH2 CH2 reversal of this sequence.
H NH H N
+ +
H NH + H NH2
C H C
H2 H2
C O C O
P P

HC + 1 HC +
N CH3 N CH3
H H

Enzyme
2
Enzyme
CH2
CH2
H O +
NH3 H NH2
H3C C COOH 3 H3C C COOH
+
H N N
CH CH
H2 H2
C O C O
P P
4
HC HC +
N CH3 N CH3
H H

5
H O O

H3C C COOH H3C C COOH


+
H NH H2N
CH CH
H2 H2
C O C O
P P

HC HC
N CH3 N CH3
H H
160 12 Amino acid metabolism

O P O

OH O
O ATP ADP O O ATP ADP
O O
C O C NH2
C O P O
H2N C O
HO O P
HO OH
1 NH3
1 OH 1 2
OH

NH2

COOH AMP CH2


CH3
COOH

CH2 A P P P CH2
NH CH2 NH NH2

HC N C CH2
HC NH2 C O AMP C O
H
COOH HC NH2
NH COOH NH NH
COOH
CH2 CH2 CH2

CH2
3 CH2 P P
3 CH2
3
CH2 CH2 CH2

HC NH2 HC NH2 HC NH2

COOH COOH COOH

4
COOH NH2 NH2

CH H2N C H2N C
H2O
CH N O NH2

COOH CH2 CH2

CH2 5 CH2

CH2 CH2

HC NH2 HC NH2

COOH COOH

Figure 12.5 Reactions of the urea cycle. Enzymes: 1, carbamoylphosphate syn-


thetase; 2: ornithine transcarbamoylase; 3: argininosuccinate synthetase; 4: argini-
nosuccinase; 5: arginase.

1. The fixation of ammonia by carbamoylphosphate synthetase, which also


uses bicarbonate as a substrate, and two molecules of ATP.
2. The transfer of the carbamoyl group from carbamoylphosphate to the
δ-amino group of ornithine, a non-standard amino acid homologous to lysine,
by ornithine transcarbamoylase. This reaction yields citrulline.
3. The ligation of aspartate and citrulline to form argininosuccinate, cat-
alyzed by argininosuccinate synthetase. This reaction again requires ATP, which
is converted to AMP in the process.
4. The cleavage of argininosuccinate into fumarate and arginine by argini-
nosuccinase.
5. The release of urea from arginine by arginase, which regenerates or-
nithine.

Urea is transported in the bloodstream and eliminated by the kidneys.


12.4 Auxiliary reactions in nitrogen transport and elimination 161

α-Keto acid 1
Amino acids 1 and 2
-
Glutamate
HCO3
NH3 α-Keto acid 2
2 α-Ketoglutarate
Carbamoyl-P
Aspartate Glutamate
Citrulline
Oxaloacetate

Ornithine Argininosuccinate Malate

Fumarate
Arginine
Urea

Figure 12.6 Cooperation of citric acid cycle, urea cycle, and transamination reactions
in urea synthesis. Typeset in bold are the names of the only metabolites for which
there is a net turnover. All other metabolites are part of closed cycles.

12.3.2 Role of aspartate in the urea cycle


As evident in Figure 12.5, only one of the nitrogen atoms in the final molecule
of urea enters the cycle as ammonia, whereas the other one is derived from
aspartate. Where does this nitrogen come from, and what becomes of the
fumarate generated from this aspartate?
To answer this question, we just need to pull together our previous knowl-
edge about the citric acid cycle and about transamination (Figure 12.6). Fu-
marate is turned into oxaloacetate in the citric acid cycle, so we can just borrow
these reactions. Oxaloacetate is then transaminated to aspartate; this gets rid
of one molecule of glutamate, which acquired its nitrogen by transamination
of another amino acid destined for degradation. In other words, the aspar-
tate serves as an intermediate carrier of nitrogen en route from amino acid
degradation to urea synthesis.

12.4 Auxiliary reactions in nitrogen transport and elimi-


nation
The urea cycle runs in the liver, yet amino acid degradation runs in many
tissues. For example, skeletal muscle has a prominent role in the degradation
of branched-chain amino acids. Therefore, a mechanism is required to carry
the nitrogen produced in the peripheral organs to the liver. Ammonia cannot
be used as a carrier since it is too toxic; amino acids are a better alternative.
The most important amino acids that serve as carriers are glutamine and
alanine. Glutamine is produced in the peripheral tissues from glutamate by
an enzyme called glutamine oxoglutarate γaminotransferase (GOGAT) or—for
162 12 Amino acid metabolism

a)
O OH O OH O OH O OH

H2N CH H2N CH C O H2N CH


NAD+
CH2 + CH2 NADH+H+ CH2 + CH2

CH2 CH2 CH2 CH2

O OH O OH O OH O NH2

b) c)
O OH O OH
COOH COOH
NAD+ NADH+H+
H2N CH C O H2N CH H2N CH

CH2 CH2 CH2 CH2


H2O NH3
CH2 CH2 CH2 CH2
H2O NH3
COOH COOH
O NH2 O OH

d) O OH O OH

ATP ADP
H2N CH H2N CH

CH2 CH2

CH2 NH3 H2O CH2

O OH O NH2

Figure 12.7 Auxiliary reactions in nitrogen transport from peripheral tissues to the
liver. a: Glutamate synthase (GOGAT) disproportionates glutamate to α- ketoglutarate
and glutamine. This happens in the periphery. b, c: Glutamate dehydrogenase (b) and
glutaminase (c) release ammonia from glutamate and glutamine, respectively. This
happens mostly in the liver. d: Glutamine synthetase scavenges excess ammonia. This
occurs both in the liver and in peripheral tissues.

the reversal of this reaction—glutamate synthase. Note that this enzyme pro-
duces an amide group, not an amine. The second product of this reaction is
α-ketoglutarate, which can participate in another round of transamination. Glu-
tamine travels to the liver, where its δ-amido group is released as ammonia by
glutaminase. The glutamate produced returns to the peripheral tissue (Figure
12.8a).3
Alanine is formed from pyruvate by transamination in the peripheral tissues.
It travels through the blood to the liver, where the nitrogen is abstracted by
transamination back to pyruvate. Gluconeogenesis then turns pyruvate into
glucose, which is returned to the periphery. This process is called the glucose
3 One might think that the glutamate could be further deaminated by glutamate dehydrogenase,

and α-ketoglutarate be returned to the periphery. However, the plasma concentration of α-


ketoglutarate is very low, so that this does not seem to be quantitatively important.
12.4 Auxiliary reactions in nitrogen transport and elimination 163

a)
Glutamine Glutamine
H 2O
3
NH3
2
Glutamate Urea
Amino acid α-Ketoglutarate

α-Keto acid Glutamate

b)

Alanine Alanine
α-Keto- α-Ketoglutarate
Amino acid
glutarate 4 5 H 2O
4 Glutamate
1
NH3
Pyruvate
Urea
α-Keto acid Glutamate Pyruvate

Glucose Glucose

Figure 12.8 Organ relationships in nitrogen transport. a: In peripheral tissues such


as skeletal muscle, glutamate is formed by transamination of amino acids being de-
graded (1). Glutamine and α-ketoglutarate are formed from two molecules of glutamate
by glutamate synthase (2; cf. Figure 12.7a). α- Ketoglutarate takes part in another round
of transamination, whereas glutamine travels to the liver. There, its amido nitrogen is
released by glutaminase (3; Figure 12.7c), and it returns as glutamate. b: The glucose-
alanine cycle combines amino acid degradation and glycolysis in peripheral tissues
with gluconeogenesis in the liver. Pyruvate formed in the periphery is transaminated
to alanine, which is transported to the liver. 4: Alanine aminotransferase; 5: glutamate
dehydrogenase.

alanine cycle (Figure 12.8b).


Nitrogen transport via glutamine does not cost any ATP, since GOGAT does
not require it. In contrast, the glucose alanine cycle is quite costly – for each
164 12 Amino acid metabolism

Portal vein / liver artery glutaminase / glutamate


dehydrogenase: NH3 ↑

NH3 is high -
urea cycle runs at speed

glutamine synthetase:
NH3↓

To systemic circulation

Figure 12.9 Distribution of enzyme activities related that release or capture ammonia
within the liver lobule. Ammonia is released from glutamine and glutamate in the
periphery of the lobule, which increases throughput of the urea cycle. Remaining
ammonia is scooped up and stored away as glutamine before the blood re-enters the
general circulation.

nitrogen transported, two ATP molecules are expended, since each round of
gluconeogenesis and glycolysis transports two nitrogens and consumes 4 mo-
lecules of ATP.

12.4.1 Auxiliary enzyme activities in urea synthesis and their distri-


bution within the liver lobule

The toxicity of ammonia requires its concentration to be kept very low in the
systemic circulation. On the other hand, for the urea cycle to run at speed,
the concentration must be high enough to saturate the initial enzyme, car-
bamoylphosphate synthetase, to a useful degree. Therefore, ammonia must
be released as the blood enters the liver tissue, and scooped up again before
the blood is drained away into the general circulation. To make this work, the
enzymes that release ammonia or fix it are strategically distributed in the liver
tissue (Figure 12.9). We had seen before that the liver has a honeycomb-like
fine structure, with the blood filtering through the individual hexagonal lobuli
from the periphery towards the center (Figure 1.10). Glutaminase and gluta-
mate dehydrogenase, which release ammonia, are found predominantly in the
periphery of the lobule. This results in an increased concentration of ammonia
in the bulk of the lobule tissue, where the urea cycle can therefore run at speed.
Scavenging the remaining ammonia is the job of glutamine synthetase, which is
mainly found around the central veins of the lobule. This illustrates nicely how
12.5 Degradative pathways of individual amino acids 165

O OH O OH

H2N CH H2N CH

CH2 CH2
H2O NH3

O NH2 O OH

Figure 12.10 The conversion of asparagine to aspartic acid by asparaginase. Note


that this is entirely analogous to the glutaminase reaction (Figure 12.7c).

biochemical and anatomical levels of organization are interrelated, and how


the body is not just a bag of cells, not even at the level of individual organs.
Glutamate dehydrogenase is credited with both release and fixation of am-
monia. Interestingly, this enzyme can utilize both NAD and NADP as cosub-
strates. The former is present in the cell mostly in its oxidized form, that is
as NAD+ , favouring release of ammonia, whereas the NADP mostly occurs as
NADPH, favouring ammonia fixation. It is not clear what regulatory mecha-
nisms prevent this from going back and forth in the same cell, which would
dissipate of the difference in the redox states of NAD and NADP.

12.5 Degradative pathways of individual amino acids

For some amino acids the degradation is a very straightforward issue. For
example, with alanine, aspartate and glutamate, transamination is all that is
required to turn them into mainstream metabolites. Glutamine and asparagine
can be accommodated by one preceding deamidation, catalyzed by glutaminase
(Figure 12.7b) and asparaginase, respectively.
Asparagine is not an essential amino acid, meaning that it can be synthe-
sized by human cells; the enzyme responsible for this, asparagine synthetase,
uses glutamine as the amide group donor. However, in some forms of leukemia,
the leukemic cells are apparently short of synthetic capacity for asparagine.
This can be exploited for therapy: Patients are treated with intravenous ap-
plication of asparaginase. 4 This lowers the serum level of asparagine and
therefore starves the leukemic cells. However, this is only one component in
the therapy – cytostatic drugs have to be added to make the therapy effective.
For the other amino acids, degradation is more elaborate. We will now consider
a few examples.

4 The enzyme is purified from the bacterium Escherichia coli. In healthy patients, injection
of a bacterial protein would rapidly induce antibodies, which would quickly render the enzyme
inactive. However, in leukemia patients, the disease itself and the cytostatic drugs simultaneously
applied conspire to suppress antibody formation. If it occurs anyway, enzyme prepared from
another bacterium, Erwinia chrysanthemi, can be used. Immunogenicity of the enzyme can be
reduced by derivatization of the protein with polyethylenglycol (PEG).
166 12 Amino acid metabolism

Figure 12.11 Alternate pathways a) O OH O OH


of serine catabolism. a: Serine dey-
dratase directly produces pyruvate H2 N CH O

by eliminating ammonia. b: Serine H2 C OH NH3 CH3

hydroxymethyltransferase removes
the side chain carbon as formalde- b)
hyde and immediately captures it H

onto tetrahydrofolic acid (THF). The H O CH2 C COOH O CH2 + CH2 COOH
remainder is glycine. c: Transamina- NH3
+
NH2
tion to hydroxypyruvate, reduction THF

to glycerate, and phosphorylation to


3-phosphoglycerate provide an alter- N,N'-methylene-THF
native shunt to glycolysis that does
not produce free ammonia.
c) O OH O OH

aKG Glu
H2N CH O

H2C OH H2C OH

NADH+H+

NAD+
O OH O OH

HC OH HC OH
H2C O P ADP ATP H2C OH

12.5.1 Degradation of serine and glycine

Both serine and glycine are non-essential amino acids, that is they can be both
synthesized and degraded in human metabolism. The formation of glycine
actually occurs in one of the degradation pathways for serine. These two amino
acids are interesting because their degradation is the prime source of single-
carbon groups in metabolism. These ‘C1 -units’ are bound to the coenzyme
tetrahydrofolic acid, which then donates them in a variety of reactions, for
example in the biosynthesis of nucleotides. For serine, there are several routes
of utilization (Figure 12.11):
1. The most straightforward one consists in its conversion to pyruvate by
serine / threonine dehydratase (Figure 12.11a). Note that in this case the nitro-
gen is released as ammonia but not by transamination. The pathway therefore
preferably occurs in the liver, which can dispose of the ammonia. The enzyme
uses the same coenzyme (pyridoxalphosphate) and a similar mechanism as the
transaminases (Figure 12.13).
2. Serine hydroxymethyltransferase cleaves serine into glycine and formalde-
hyde. Formaldehyde, which is highly reactive, is not released by the enzyme
but is immediately fixed onto the cosubstrate tetrahydrofolic acid.
3. An alternative pathway transaminates serine to hydroxypyruvate, which
is then reduced to glycerate and phosphorylated to 3-phosphoglycerate. While
both pyruvate and 3-phosphoglycerate can serve as substrates for gluconeoge-
12.5 Degradative pathways of individual amino acids 167

a)
Enzyme Enzyme Enzyme

CH2 CH2 CH2


+
NH2 H H2N H NH2

H O CH2 C COOH H2C COOH


O CH2 HC COOH
+ +
HN + HN
HN
CH CH
CH H2
H2
H2
C O C O
C O
P P
P
HC HC
+ HC +
N CH3 N CH3
H N CH3 H
H
H2O

E-PLP
H2N CH2 COOH

b) H
H 2N N N

N
N O COOH O
H
OH N C N C C C C OH
H H H H2 H2
n

H H
N N

N N

H N N
CH2 CH2
O H HO H

H
H2N N N

N
N O COOH O

OH H2C N C N C C C C OH
H H H2 H2
H2O n

Figure 12.12 Catalytic mechanism of serine hydroxymethyltransferase. The reaction


proceeds in two steps. a: PLP-dependent cleavage of serine to glycine and formalde-
hyde. b: Capture of formaldehyde by tetrahydrofolic acid (THF). In the resulting N,N’-
methylene-THF, the mobilizable methylene group is held by two nitrogen atoms as by
a pair of tweezers.

nesis, this pathway avoids the release of free ammonia.


It is interesting to note that all the three routes of serine degradation in-
volve enzymes that contain pyridoxalphosphate (PLP) as a coenzyme. This is
obvious for the third route, since it starts out with transamination. For serine
dehydratase and serine hydroxymethyltransferase, this is illustrated in Figure
12.12 and Figure 12.13. Please stare at these figure and at Figure 12.4 until you
notice the similarities in the roles of PLP in all three reactions. In all cases, the
reversible transfer of an electron to the coenzyme destabilizes the substrate
168 12 Amino acid metabolism

Enzyme
Enzyme Enzyme
CH2
CH2 CH2
NH2
+
NH2 NH2 H2O
H H
HO CH2 C COOH H2C C COOH
HO CH2 C COOH
+ +
HN + HN
HN
CH CH
CH H2
H2
H2
C O C O
C O
P P
P
HC HC
+ HC +
N CH3 N CH3
H N CH3 H
H
H2O

+
H E-PLP
NH3 H2O

H3C C COOH H3C C COOH H2C C COOH

O NH NH2

Figure 12.13 Catalytic mechanism of serine dehydratase. While the overall reaction
consists in the elimination of ammonia, the enzyme itself actually eliminates water,
which is reflected in its name. The aminoacrylate released by the enzyme is then
spontaneously hydrolyzed to pyruvate and ammonia.

and primes it for the reaction; the specific bond within the substrate that is
broken is then determined by the point of attack of some specific side chains
within each enzyme’s active site.
We have already seen that glycine is a product of serine hydroxymethyl-
transferase. Since that enzyme reaction is reversible, glycine can actually be
converted to pyruvate or 3-phosphoglycerate via serine and thus serve as a
substrate in gluconeogenesis. The required additional carbon, in the form of
N,N’-methylene-THF, can be provided by the glycine cleavage system, an enzyme
that completely breaks down glycine (Figure 12.14). Therefore, two glycines
are required to provide one pyruvate. The glycine cleavage system again uses
pyridoxalphosphate in its catalytic mechanism, and as far as I can see the PLP
again functions in a similar way as described before.

12.5.2 Degradation of leucine

Leucine, isoleucine and valine are collectively referred to as the branched-chain


amino acids. One special thing about these is that their degradation proceeds
largely in skeletal muscle. This is similar to the degradation of fatty acids,
which also occurs to a large extent in muscle, and indeed several steps in
leucine degradation have similarity with the reactions we have seen in fatty
acid metabolism. Leucine degradation involves the following reactions (Figure
12.15):
12.5 Degradative pathways of individual amino acids 169

glycine THF serine pyruvate

NAD+ NH3

NADH + H+
methylene-
NH3+CO2 glycine
THF
Figure 12.14 Glycine catabolism. The glycine cleavage system completely breaks
down glycine, extracting one methylene group as N,N’-methylene-tetrahydrofolate. This
methylene group can be used by serine hydroxymethyltransferase to convert a second
glycine molecule to serine and then to pyruvate.

1. Transamination by branched chain amino acid (BCAA) transaminase yields


α-ketoisocaproate.
2. α-Ketoisocaproate undergoes decarboxylation and dehydrogenation by
BCAA dehydrogenase. Like the transaminase in step 1, this dehydrogenase is
active with all branched chain amino acids (valine, leucine, isoleucine). The re-
action and the organization of the enzyme is completely analogous to pyruvate
dehydrogenase and α-ketoglutarate dehydrogenase, and all use the very same
E3 subunit.
3. The resulting metabolite, isovaleryl-CoA, is similar in structure to a fatty
acyl-CoA. It likewise undergoes a FAD-dependent dehydrogenation reaction (by
isovaleryl-CoA dehydrogenase), which yields isopentenyl-CoA.
4. Biotin-dependent carboxylation yields methylglutaconyl-CoA. In all of the
previous carboxylation reactions we saw, a vicinal carbonyl group facilitates
carboxylation by withdrawing electrons from the carbon that gets carboxylated.
This electron-withdrawing effect can be relayed by a conjugated C− −C double
bond, which is the case here.5
5. Addition of water by methylglutaconyl-CoA hydratase yields HMG-CoA.
6. HMG-CoA is split by HMG-CoA lyase to acetyl-CoA and acetoacetate, as
seen before in ketone body synthesis. Evidently, leucine is a purely ketogenic
amino acid.

12.5.3 Degradation of phenylalanine


While the degradation of leucine shows a comforting similarity to previously
encountered pathways, the situation is profoundly different with phenylalanine.
This is due to the aromatic nature of the side chain. Aromatic rings are quite
stable, and therefore some brute force is needed to crack them open. The best
tool for this task is molecular oxygen, and lots of it is used in the breakdown
of phenylalanine.
5 The mediation of electron-withdrawing effects by conjugated C=C double bonds is referred

to as the vinylogous effect.


170 12 Amino acid metabolism

COOH COOH CoA CoA


α-KG NAD+ FAD
H2N CH C O S S
Glu NADH+H+ FADH2
CH2 CH2 C O C O

C C CH2 CH
H3C H CH3
1 H3C H CoA 2 CO2 3
CH3
C
H3C H H3C CH3
CH3

CO2
CoA CoA CoA 4
H2O ATP
S S S

C O C O C O
ADP+Pi
CH3 6 CH2
5 CH

H3C C C COOH HO C C COOH


H2 H2 H3C C COOH
O CH3 H2

Figure 12.15 Degradation of leucine. The reactions are numbered in accordance with
the descriptions in the text.

The degradation of phenylalanine involves the following reactions (Figure


12.16):
1. The hydroxylation of phenylalanine to tyrosine by phenylalanine hydrox-
ylase. In this reaction, the second oxygen atom is released as water, reduced at
the expense of the redox cosubstrate tetrahydrobiopterin.
2. Transamination of tyrosine yields p-hydroxyphenylpyruvate.
3. Advanced magic that uses another oxygen molecule (and ascorbic acid,
vitamin C, as a cofactor) turns hydroxyphenylpyruvate into homogentisate. I
have traced the bits and pieces in this reaction by highlighting them in color
or boldface, but an actual explanation of the mechanism is beyond my feeble
powers of understanding and narration.
4. Ring cleavage involves a third molecule of oxygen. The product is maley-
lacetoacetate.
5. Maleylacetoacetate is cis-trans isomerized to fumarylacetoacetate.
6. Fumarylacetoacetate is hydrolyzed to fumarate and acetoacetate.
Fumarate can be utilized in gluconeogenesis, whereas acetoacetate cannot.
Whether this means that phenylalanine is both glucogenic and ketogenic, or
just glucogenic because it can contribute to gluconeogenesis, is a purely verbal
question that we will leave unanswered.

12.6 Hereditary enzyme defects in amino acid metabolism


Since there are as many pathways as there are individual amino acids, it is
understandable that many of the known inborn errors of metabolism are related
to amino acid metabolism. We will consider but a few examples that affect the
pathways discussed here.
12.6 Hereditary enzyme defects in amino acid metabolism 171

COOH COOH Figure 12.16 Degradation


H2N CH
Biopterin-H4 H2N CH of phenylalanine. Enzymes:
Biopterin-H2 1, Phenylalanine hydroxylase;
CH2 CH2
2, tyrosine transaminase;
3, p-phenylpyruvate dioxy-
HC CH HC CH genase; 4, homogentisate
HC CH
1 H 2O HC CH dioxygenase; 5, maleylace-
C O2 toacetate isomerase; 6,
H
α-KG OH fumarylacetoacetase.

2
COOH
Glu
C O

OH COOH CH2

CH2 CO2 H 2O O2
HC HC CH

HC CH
3 HC CH

OH O2 Ascorbate-H2 OH

4 Ascorbate

OH
COOH
O O O
O
HC CH2 HO H
O C C C C
C C C OH
HC H H2 H2
CH2
C 5 O

O
6 H 2O

O O O
H
HO C C H C
C C OH C C OH
H H2 H2
O

12.6.1 Phenylketonuria

Phenylketonuria is the most common hereditary enzyme defect, at least among


Europeans. It is clinically manifest in about one among ten thousand persons.
Considering that only homozygous q people are clinically affected, this works
4 1
out to a heterozygote frequency of 10000 = 50 , that means one in fifty persons
carries a copy of the defective gene and can potentially have children with this
disease.
The enzyme affected is phenylalanine hydroxylase, the first enzyme in the
degradative pathway (Figure 12.16). The name of the disease stems from the
fact that phenylpyruvate and some derivatives thereof are found in the urine.
Formation of phenylpyruvate is due to the buildup of phenylalanine, which will
172 12 Amino acid metabolism

COOH
Phenylpyruvate C O

CH2

HC CH

HC CH
Tyrosine- C
H
aminotransferase

Phe Tyr Hydroxyphenyl- neurotoxic


pyruvate metabolites
Phenylalanine
hydroxylase

Figure 12.17 Metabolic derailments in phenylketonuria. See text for details.

eventually cause it to overcome the high KM of tyrosine transaminase (Figure


12.17). Phenylpyruvate is believed to give rise to neurotoxic metabolites, al-
though the exact nature of these metabolites is not clear. Symptoms include
retardation of neurological and mental development.
The treatment of phenylketonuria is pretty straightforward: Limitation of
dietary phenylalanine. Tyrosine is plentifully available in a reasonably protein-
rich diet, so that the lack of endogenous formation won’t be a problem. The
challenge then is to diagnose the disease in newborn kids, before any damage
is done. Happily, the enzyme defect does not cause a problem during fetal
development, since both useful and potentially harmful metabolites are con-
stantly equilibrated between the maternal and the fetal circulation. Buildup
of a metabolite in the fetus will therefore not occur as long as the mother’s
metabolism is able to degrade it.
The modern test for phenylketonuria is boring: Just draw a sample of blood
and determine the phenylalanine concentration in the serum by HPLC. The
original test—the Guthrie test—was a bit more roundabout in principle, yet
ingenious, exceedingly simple and cheap in practice. Moreover, it nicely illus-
trates the power of bacterial genetics in biochemistry, and it therefore merits
inclusion here.
In contrast to mammalian cells, the bacterium Escherichia coli can synthe-
size all 20 standard amino acids, as long as it has ammonia, some inorganic
salts, and an organic carbon source such as glucose. Such a mixture constitutes
a minimal medium. The Guthrie test makes use of a mutant E. coli strain that
is Phe – , which means that it cannot synthesize phenylalanine on its own. This
strain can be grown on a medium that supplies phenylalanine; however, on
minimal medium, it will not grow. Here is how it works (Figure 12.18):
1. A petri dish with minimal medium is streaked with the Phe- strain. The
cells will be thinly spread everywhere, but they will not grow for lack of pheny-
lalanine.
12.6 Hereditary enzyme defects in amino acid metabolism 173

Minimal medium (no phenylalanine): Rich medium (contains phenylalanine):


No growth of Phe- strain Growth of Phe- strain

Spread Phe- strain evenly on the


surface of minimal medium:
No growth will occur unless..

... we place a our test strips


soaked with blood onto the
agar. Phe diffuses into agar…

… and creates a zone of


bacterial growth around the
sample from an afflicted child

Figure 12.18 Principle of the Guthrie test for phenylketonuria. See text for additional
details.

2. A small piece of filter paper, soaked with a drop of test blood, is placed
on the surface of the agar.
3. Small molecules including amino acids will diffuse from the filter paper
into the surrounding agar. If the test sample contains a high level of pheny-
lalanine, as is the case with infants that suffer from phenylketonuria, bacterial
cells right next to the filter paper will gobble it up and start growing.
4. If very little phenylalanine is available, as will be the case with healthy
children, no bacterial growth will occur around the samples.
Therefore, a zone of bacterial growth around the filter paper snippet will
identify a sample from a patient with phenylketonuria. Note that, for this test
to work, we cannot collect the blood sample right away after birth. In the
womb, the fetal blood equilibrates with the mother’s, and so the phenylalanine
174 12 Amino acid metabolism

concentration is only slightly increased (which is good, since it protects the


fetus from the damage). We therefore have to allow about 1-2 weeks after
delivery for phenylalanine to accumulate in the child’s blood. This is one of
the drawbacks of the Guthrie test in comparison to the HPLC method. The
latter is more quantitatively accurate and readily detects the smaller increase
in phenylalanine concentration observed at the time of delivery.

12.6.2 Tyrosinemia type I


This tyrosinemia is another metabolic defect in the degradation of phenylala-
nine and tyrosine. It is very common in parts of Quebec, which is probably re-
lated to the original immigration of a comparatively small group of settlers. The
deficient enzyme is fumarylacetoacetase. All preceding metabolites are backed
up (Figure 12.19a). Severe liver damage is prominent among the symptoms;
this is ascribed to the toxic effect of maleylacetoacetate, which reacts with DNA
and thereby causes damage to it. Therapy is possible with the enzyme inhibitor
nitro-trifluoromethyl-benzoyl-cyclohexanone (Figure 12.19b). This compound
is structurally similar to both ascorbic acid and p-hydroxyphenylpyruvate, the
cosubstrate and the substrate of p-hydroxyphenylpyruvate dioxygenase. Inhi-
bition of this enzyme relieves backup of all toxic downstream metabolites. To
reduce the elevated concentration of tyrosine, which can still lead to neurologi-
cal deficits, dietary restriction of that amino acid and of phenylalanine is also
required.
12.6 Hereditary enzyme defects in amino acid metabolism 175

a) Tyrosine ↑

p-Hydroxyphenylpyruvate ↑

Homogentisate ↑

Maleylacetoacetate ↑ Toxic effect


(liver damage)
Fumarylacetoacetate ↑

fumarylacetoacetase
Fumarate + acetoacetate

b) Tyrosine ↑

p-Hydroxyphenylpyruvate ↑

p-Hydroxyphenylpyruvate dioxygenase
Homogentisate O O NO2

Maleylacetoacetate
O CF3
Fumarylacetoacetate
(Nitro-trifluoromethyl-benzoyl)-
cyclohexanone (NTBC)
Fumarate + acetoacetate
Figure 12.19 Tyrosinemia type I. a: Enzyme defect and resulting metabolic backup
in in tyrosinemia type; b: Therapy with the enzyme inhibitor nitro-trifluoromethyl-
benzoyl-cyclohexanone (NTBC).
Chapter 13

Hormonal regulation of metabolism

Metabolism is regulated at different organizational levels. With regulatory


mechanisms that operate entirely within the individual cell, the main purpose is
housekeeping, that is to adjust the throughput of catabolic pathways such as to
keep up a ready supply of ATP, NADPH and so on. This is accomplished largely
by feedback inhibition and feedforward activation by key metabolites such as
ATP and acetyl-CoA. In contrast, hormonal control is about the obligations of
a cell to the organism as a whole. Hormones are released in response to the
prevailing metabolic situation of the body. Key examples are the secretion of
insulin in response to high blood glucose levels, and the secretion of glucagon in
response to low glucose levels. Hormones may also be secreted in anticipation
of an imminent change of the metabolic situation, as is the case with the ’fight-
and-flight’ hormones epinephrine and norepinephrine. Other hormones with
major effects on energy metabolism are the glucocorticoids (cortisone and
hydrocortisone) and the thyroid hormones (tri- and tetraiodothyronine).

13.1 Insulin
The insulin most widely known hormone in metabolic regulation is certainly
insulin, which facilitates the utilization of glucose. This hormone is so named
because it is secreted in the pancreatic islets (insula = latin for island, islet).
These islets are small portions of tissue that are found interspersed within the

176
13.1 Insulin 177

a) Figure 13.1 Pancreatic islets, and the


purification of insulin from them. a: An
islet (marked by arrows) within the ‘red
sea’ of exocrine pancreas. Reproduced,
with permission from Roger Wagner,
from http://www.udel.edu/Biology/
Wags/histopage/histopage.htm.
b: Initial attempts to purify insulin from
pancreas homogenates failed, because
the hormone was rapidly degraded by
the proteases released from the exocrine
b) cells. c: Banting and Best managed
to purify insulin by first occluding
Trypsin, Insulin the pancreatic ducts of experimental
animals. This led to the degeneration of
the exocrine pancreas, while the islets
Garbage were left intact. d: Frederick Banting
(left) and Charles Best.

c)

d)

sea of the exocrine pancreas. The exocrine1 pancreas, which accounts for 98%
of the entire mass of the organ, is responsible for secreting all the enzymes and
bicarbonate we have encountered before. The islets, in contrast, are endocrine
and secrete a variety of peptide hormones into the blood stream, most notably
insulin and glucagon.
The importance of the pancreas in the regulation of blood glucose was rec-
1 Exocrine means that the secretion is targeted to the ’outside’ world (and that would include

the intestinal lumen), whereas endocrine secretion is targeted into the blood stream. Thus, all
hormones are secreted by endocrine glands, and the branch of medicine that specializes in
hormones, their glands and related diseases is endocrinology.
178 13 Hormonal regulation of metabolism

Figure 13.2 Primary struc- N-GIVQQCCASVCSLLYQLENYCN-C


ture of insulin. Top: Amino
acid sequences from cow, pig, N-FVNQHLCGSHLVEALYLVCGERGFFYTPKA-C
and humans. Deviations of
the animal sequences from
the human one are high- N-GIVQQCCTSICSLLYQLENYCN-C
lighted. The lines indicate
N-FVNQHLCGSHLVEALYLVCGERGFFYTPKA-C
disulfide bonds; one disul-
fide bond occurs within the
(shorter) A chain, whereas A N-GIVQQCCTSICSLLYQLENYCN-C
and B chain are held together
by two disulfide bonds. Bot- N-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-C
tom: Processing of insulin
precursors. The two final
products (insulin and C pep- Signal
tide) are both stored in secre- peptide
tory vesicles and secreted to-
gether.

Preproinsulin Proinsulin C-peptide Insulin

ognized by animal experiments, which showed that removal of the pancreas


resulted in diabetes. Attempts to purify the active principle—that is, insulin—
failed, however, since after grinding up entire pancreas organs all proteins were
rapidly degraded by the proteases released from the exocrine pancreas cells
(Figure 13.1a). The bright idea that solved this problem occured to Dr. Fred-
erick Banting of the University of Toronto (Figure 13.1d). He took advantage
of the previous observation that obstruction of the pancreatic duct induces
self-digestion and destruction of the exocrine pancreas, however somewhat
surprisingly without destruction of the islets. By first occluding the pancreatic
duct in the animals and then patiently awaiting the degeneration of the exocrine
pancreas, Banting and his student Charles Best were able to purify insulin in
intact form and demonstrate its ability to revert diabetic symptoms. In remark-
able contrast to modern practice, they very generously refrained from claiming
a patent. Instead, they invited everyone to join in and supply the world with
insulin, so that within a short period of time it became available and affordable
to diabetics worldwide. One simply must love these guys. Great Canadians, and
of course well-deserved Nobel prize winners.2
Insulin became the subject of a second Noble prize when Frederick Sanger at

2 Charles Best did not officially receive the Nobel Prize; instead, Banting shared it with John

MacLeod, another researcher at U of T. However, Frederick Banting declared that Best should
have been included and split his share of the prize with him to affirm this point. Thanks to
Alastair Brownie for correcting this historical detail.
13.1 Insulin 179

Cambridge managed to develop the first workable methodology for protein se-
quencing and to elucidate the primary structure of insulin, which thus became
the first protein with completely known amino acid sequence. The primary
structure of insulin is depicted in Figure 13.2. It consists of an A chain and a
B chain, which are derived from a common precursor that undergoes a series
of site-specific cleavages. The final products, insulin and the C peptide, are
both stored in secretory vesicles and are released together. Note the sequence
variations between the bovine, the porcine and the human insulin. Because
of these sequence variations, it is possible for diabetics to develop antibodies
against the animal insulins; this is a major reason why nowadays recombinantly
produced human insulin is preferred for the treatment of diabetes.

13.1.1 Mechanism of secretion of insulin from pancreatic β-cells


Insulin is secreted by a certain cell type within the pancreatic islets, the β-cells
(α-cells secrete glucagon, the antagonist hormone of insulin). The secretion is
controlled by the level of glucose in the blood. The textbook model for this
regulation works as follows:
1. Glucose enters the β-cell and gets degraded, which yields ATP. Thus, the
level of intracellular ATP varies in proportion to the blood glucose.
2. ATP binds to the sulfonylurea receptor, which is associated with the
Kir K+ channel. Binding of ATP closes the channel, which depolarizes the
membrane.
3. Depolarization opens voltage-gated Ca++ channels and allows Ca++ into
the cell. The increased intracellular Ca++ then triggers insulin secretion by
exocytosis.This step is analogous to neurotransmitter exocytosis in nerve cells
in response to presynaptic action potentials.
The sulfonylurea receptor is so named because it is the target of sulfony-
lurea derivatives such as tolbutamide (Figure 13.3). These drugs mimic the
effect of ATP on the receptor, thus stimulating the release of insulin from the
β-cells. This is an important therapeutic principle in diabetes type II. We will
learn more about this later.

13.1.2 The insulin receptor and its intracellular second messengers


and effectors
The biochemical effects of insulin on its target cells can be summarized in one
simple phrase: Obscenely complex. The intention of the following is to just give
you a feel of that complexity, but there is no obligation to memorize each and
every detail of it. The insulin receptor is a transmembrane protein located in the
cytoplasmic membrane. It has an extracellular domain that specifically binds
insulin; binding causes activation of the intracellular domain, which is a protein
tyrosine kinase (Figure 13.4a). The first protein that gets phosphorylated by
this enzyme is another insulin receptor molecule (Figure 13.4b). This mutual
180 13 Hormonal regulation of metabolism

Figure 13.3 The control of a) Insulin


Glucose
insulin secretion. a: Overview.
b: Structural sketch of the
Ca++
sulfonylurea receptor (green)
and the associated Kir K+
channel. The cylinders repre- Glucose

+
sent transmembrane helices. Insulin
ADP+Pi
The ATP binding folds (ABF) Membrane
are on the cytosolic side of potential
K+
the membrane. c: Binding
of ATP will cause a confor- ATP
H2O + CO2
mational change to the sul-
fonylurea receptor, which in
turn will cause the channel to
close. This will partially de-
polarize the membrane and b) Kir-Channel Sulfonylurea receptor
induce insulin secretion via
opening of a voltage-gated
calcium channel. d: Structure
of tolbutamide, an oral drug
that binds to the sulfonylurea
receptor and is used in type
II diabetes. The sulfonylurea ABF ABF
moiety is highlighted.

c)

K+

ATP ATP

ATP

O O
d)
H3C S N N C C C CH3
H H H2 H2 H2
O

phosphorylation of insulin receptors locks them in the active state, from which
they will only revert to the inactive state upon dephosphorylation by a protein
tyrosine phosphatase, even if insulin leaves the receptor again.3
Apart from this mutual phosphorylation, the insulin receptor kinase acts
3 However, it seems that insulin stays bound to the receptor for very long periods of time.
13.1 Insulin 181

a) Ligand

Receptor domain
(extracellular)

Enzyme domain
(intracellular)

Inactive enzyme Active enzyme

b)

ATP ADP
P P
P P

IRS-1 IRS-1 P
ATP ADP P
P

IRS-2(3,4) IRS-2(3,4) P
ATP ADP P

Figure 13.4 The insulin receptor. a: The receptor is a transmembrane protein that
has an extracellular ligand-binding domain and an intracellular protein kinase domain;
the kinase is activated by binding of insulin. b: Ligand-activated insulin receptors will
first perform mutual phosphorylation and then phosphorylate a series of intracellular
regulatory proteins, the so-called insulin receptor substrates (IRS).

upon number of intracellular proteins, which are collectively referred to as in-


sulin receptor substrates (IRS; see again Figure 13.4b). In their phosphorylated
states, these proteins activate a variety of other so-called adapter proteins,
which then control various effector cascades. Some important regulatory cas-
cades are shown in Figure 13.5:
1. The stimulation of glycogen synthesis. Figure 13.5a shows the cascade
182 13 Hormonal regulation of metabolism

that leads to the activation of glycogen synthase:


(a) The receptor phosphorylates insulin substrate 1 (IRS-1).
(b) Phosphorylated IRS-1 binds to and activates PIP2 -3-kinase, which phospho-
rylates the membrane lipid phosphatidylinositol-bisphosphate (PIP2 ) to the
corresponding trisphosphate (PIP3 ).
(c) PIP3 binds to and activates protein kinase B, which in turn phosphorylates
glycogen synthase kinase 3.
(d) In its phosphorylated state, glycogen synthase kinase 3 is less active, which
results in a lower degree of phosphorylation and therefore higher activity
of glycogen synthase.
Note that this is only one of several regulatory mechanisms by which insulin
affects glycogen synthesis. Another one consists in the activation of phospho-
diesterase, which will lower the level of cAMP, thereby countering the effects of
epinephrine and of glucagon (see later).
2. The induction of enzymes for glycolysis. This cascade is shown in Figure
13.5b:
(a) The first intermediate is again IRS-1. However, in this case IRS-1 it binds
to a different adapter protein, which is called Grb-2.
(b) Grb-2 forms a complex with two other proteins, Sos and Ras; this results in
the exchange of GDP bound to Ras for GTP, and subsequently the activation
of a protein kinase called Raf-1.
(c) Two more protein kinases—MEK and MAPK—are phosphorylated in turn.
The latter one acts on nuclear transcription factors, which then increase
the rate of transcription of the genes encoding enzymes for glycolysis and
for triacylglycerol synthesis.
3. The increase of cellular glucose uptake. This is accomplished by the
translocation of GLUT4 transporters from intracellular vesicles to the cytoplas-
mic membrane. This translocation is reversible (Figure 13.5c). The cascade
controlling this translocation again involves IRS-1, PIP3 , and protein kinase B
(Figure 13.5d).
One question that often comes to mind in this context is: Why did nature
invent all these complicated, lengthy cascades? The standard textbook answer
is signal amplification: If each molecule of protein kinase X activates multiple
molecules of kinase Y, each of which in turn activates multiple molecules of
effector enzyme Z, then the number of final effector molecules can be far
larger than the number of hormone molecules that did bind to the cell. A
valid example for this rationale is glycogen phosphorylase. However, signal
amplification is not always a compelling reason. For example, the number
of nuclear transcription factor molecules required to trigger transcriptional
changes is probably not any larger than the number of insulin receptors on
the cell surface, so no signal amplification is needed in this case. What other
reasons could there be?
13.1 Insulin
a) I I c)
Glucose
I I Glucose transporter 4
exposed on cell surface
P-Glycogen synthase cytoplasmic
PP (inactive)
membrane

Glycogen synthase Glucose


PI-3K P IRS-1 IRS-1 PKA High insulin Low insulin
kinase 3 (active)

Glycogen synthase Glucose transporter 4


PIP2 PIP3 PKB (active) stored in vesicles

P-Glycogen synthase
kinase 3 (inactive)

b) I I d)
I I
I I
I I
Transport of GluT4 to cell
surface by vesicle fusion
PP Phosphorylation of nuclear factors,
induction of genes for glycolytic PP
enzymes etc.
IRS-1 IRS-1 P Grb-2
PI-3K P IRS-1 IRS-1
Sos MAPK-1-P
GTP
displaces Phosphorylation of
Ras MEK-P PIP2 PIP3 PKB
GDP cytoskeletal proteins

Raf-1 MAPK-1
MEK

Figure 13.5 Intracellular effects of insulin receptor stimulation. a: One of the pathways that lead to activation of glycogen
synthesis. b: Mediation of transcriptional effects of insulin. c: Translocation of glucose transporters from intracellular vesicles
to the cytoplasmic membrane. d: Movement of glucose transporters is mediated by protein phosphorylation, too. (See text for
further details.)

183
184 13 Hormonal regulation of metabolism

Ligand

extracellular space N

‘7TM’ - receptor

cytosol
γ β α GTP
γ β α heterotrimeric
G-protein
GDP
GDP

N N
inactive active

γ β α Pi γ β α
GDP GTP

Figure 13.6 Function of a G protein-coupled receptor. Upon binding of the ligand to


the extracellular face, a conformational change occurs to both the receptor and the G
protein bound to it. This leads to the exchange of GDP for GTP on the α-subunit of
the G protein, which then dissociates from the βγ-dimer and finds its effector protein.
When the GTP’ase activity that is built into the α-subunit cleaves GTP to GDP, the α-
subunit reverts to its inactive conformation, leaves its target protein, and re-associates
with the β- and γ-subunits and the receptor.

I personally prefer the idea that this complexity serves the purpose of allow-
ing different signaling pathways to interact and still yield one coherent, sensible
response. The network of intracellular kinases, phosphatases and adapter pro-
teins constitutes the brain of the cell, and a brain just needs a certain level of
complexity.

13.2 Glucagon and epinephrine


The metabolic effects of glucagon epinephrine are similar to each other but
opposite to that of insulin. Glucagon is mainly concerned with metabolic reg-
ulation, whereas epinephrine also has pronounced effects on heart rate, blood
pressure and so on. While the two hormones have separate, specific receptors,
both of these fall into the group of the G protein-coupled receptors (GPCRs).
The workings of a GPCR are illustrated in Figure 13.6. The receptor is a mem-
brane protein with seven transmembrane helices, which on its cytosolic face
is associated with a GTP-binding protein, or G protein. This protein has three
subunits; the α-subunit is bound to GDP in its resting state. Signal transduction
by a GPCR works as follows:
1. Binding of the ligand to the extracellular face of the receptor causes a con-
formational change that involves both the receptor and the heterotrimeric
G protein.
2. On the G protein, the conformational change leads to the exchange of
GDP for GTP on the α-subunit.
13.2 Glucagon and epinephrine 185

Adenylate cyclase

α
ATP
GTP γ β

cAMP cAMP
R R cAMP R R
cAMP cAMP
C C

Protein kinase A,
Protein kinase A, inactive C C active

Figure 13.7 Activation of adenylate cyclase and protein kinase A by epinephrine or


glucagon. The α-subunit of the G protein binds to adenylate cyclase, which causes it
to synthesize cAMP. Protein kinase A, in its resting state, exists as a complex of two
catalytic (C) and two regulatory (R) subunits. Binding of cAMP to the R subunits releases
and thereby activates the catalytic subunits.

3. Upon binding of GTP, the α-subunit dissociates from the βγ-dimer and
binds to its effector protein.
4. After a certain amount of time, the GTPase activity that is built into the
α-subunit cleaves GTP to GDP.
5. Cleavage of GTP causes the α-subunit to revert to its inactive conforma-
tion, to leave its target protein, and to re-associate with the βγ-dimers.
The system thus returns to its resting state.
Both the epinephrine receptor More specifically, the β-adrenergic receptor,
of which there again are several subtypes. and the glucagon receptor are cou-
pled to the same G protein, which binds to adenylate cyclase as its effector
protein and activates itadenylate cyclase. Because of this, the metabolic effects
on a cell that has receptors for both of them will always be similar. Differences
between the effects of the two hormones arise from the fact that the receptors
have different tissue distributions; many cells have receptors for epinephrine
but not glucagon.
The consequences of the stimulation of adenylate cyclase are depicted in
Figure 13.7. Protein kinase A, as we have seen before, has several effects:
1. It phosphorylates and thereby inactives glycogen synthase.
2. It also phosphorylates phosphorylase kinase, which in turn phosphory-
lates and thereby activates glycogen phosphorylase, so that more glucose gets
released from glycogen.
186 13 Hormonal regulation of metabolism

COOH
Figure 13.8 Nuclear a)
H2 N CH
hormone receptors and
CH2
transcriptional regulation. CH2OH

C O
a: Structures of cortisol (left) CH3
HO OH
and of the thyroid hormone I I
triiodothyronine (right). The CH3
O
iodines contained in this H H

hormone (and the similar O

tetraiodothyronine) are the I

reason why you need iodine OH

in your diet. b: Simplified


schematic of the common b) H
mode of function of the two
hormones. H: hormone; R:
Proteins
receptor. Of course, there are
(Enzymes, Receptors, …)
different, specific receptors
for the two hormones. R
H H
R

mRNA

3. It phosporylates phosphofructokinase 2, thereby switching on the fructose-


2,6-bisphosphatase activity on that bifunctional enzyme. This reduces the
concentration of fructose-2,6-bisphosphate and slows down glycolysis and ac-
celerates gluconeogenesis.
4. It phosphorylates and thereby activates hormone-sensitive lipase in fat
tissue. This leads to the release of glycerol and fatty acids into the blood.
As stated before, all these effects are antagonistic to those of insulin and
will tend to sustain or increase blood glucose levels.

13.3 Glucocorticoids and thyroid hormones


Glucagon and epinephrine act primarily on the activity of pre-formed enzymes
(protein kinase A affects transcriptional regulation as well, though). Insulin
both affects pre-formed enzymes and induces the synthesis of new ones. Glu-
cocorticoids thyroid hormones mostly act by way of genetic regulation. Their
receptors are not located at the cell surface but inside the cell. They are either
found in the nucleus to begin with, or they translocate from the cytosol to
the nucleus upon binding their ligand. The mechanism of this regulation is
depicted in Figure 13.8 in simplified form. Binding of the hormone (H) to the
receptor (R) causes the latter to recruit a bunch of other proteins (omitted) and
together with these bind to specific target sequences in the DNA. This changes
the expression levels of genes nearby. The proteins encoded by these genes will
include enzymes of energy metabolism but also other hormone receptors; for
13.4 Leptin 187

Figure 13.9 Obese mice, which are homozygously deficient for leptin, can be brought
to normal weight by way of leptin substitution. (Image ripped off from http://www.
biochemsoctrans.org/bst/033/1063/bst0331063f01.gif.

example, cortisol and cortisone induce enzymes for glycogen metabolism and
gluconeogenesis but also receptors for epinephrine. Major effects of cortisol
action are:
1. Stimulation of protein breakdown. The amino acids released are largely
used for gluconeogenesis.
2. Increased enzyme activities for glycogen synthesis and breakdown (!), and
for gluconeogenesis.
3. Increased sensitivity to epinephrine and norepinephrine.
4. Suppression of pain and inflammation by inhibition of prostaglandin syn-
thesis.
Cortisol is often considered a chronical stress hormone, in contrast to the
acute stress hormones epinephrine and norepinephrine.
Thyroid hormones also have a variety of regulatory targets. One key effect is
the induction of thermogenin, which is simply a respiratory chain uncoupling
protein. The mode of action of uncoupling proteins has been described in
section 6.1. The up-regulation of uncoupling proteins is responsible for the
finding of accelerated metabolism, hyperthermia and weight loss in patients
with excessive thyroid hormone secretion (hyperthyreosis).

13.4 Leptin
A relatively recently discovered hormone is leptin. This peptide hormone is
released by fat cells and taken up by the hypothalamus, where it restricts
the appetite. The hypothalamus has a key role in the homeostasis of many
physiological parameters and controls the autonomic nervous system as well
as the hypophyseal gland, which in turn controls the activity of the thyroid and
adrenal glands. The major variable that controls leptin secretion is simply the
volume of fat tissue, which means that leptin regulates metabolism on a much
longer time scale than the other hormones discussed above.
The discovery of leptin was accomplished with two different mice strains
that lack the hormone, named “obese mice”, or the receptor, named “diabetic
188 13 Hormonal regulation of metabolism

mice”. Despite these names, both strains show the same combination of over-
weight and type II diabetes. While the receptor-deficient strain unsurprisingly is
resistant to leptin, the hormone-deficient one reacts to the hormone in a quite
impressive fashion (Figure 13.9. Leptin deficiency is not a common condition
in humans, however, so that the therapeutic potential of leptin substitution is
limited.
Chapter 14

Diabetes mellitus

To conclude this course, we will try and apply what we have learned to un-
derstand the metabolic phenomena underlying the most common metabolic
disease, diabetes mellitus.
The name ‘diabetes mellitus’ means ‘honey-sweet flow-through’.1 That tells
us about two things:
1. An important symptom of the disease: Copious flow of urine, containing
significant amounts of glucose.
2. A classical method of diagnosis. The alternative to diabetes mellitus is
diabetes insipidus (insipid, flavourless), which is caused by a deficiency of the
antidiuretic hormone due to lesions in the posterior lobe of the hypophyseal
gland and is not related to glucose metabolism.
If you plan on going on to medical school, you will be glad to learn that the
classical method of diagnosis is no longer used in clinical practice.
1 For geeks: More precisely, ‘marching through’ – you may recall the term ‘adiabatic’ from

thermodynamics, meaning ‘without permeation’, which is the opposite of ‘diabetic’. The syllable
also occurs in the title of Xenophon’s work Anabasis, or Invasion, which details his experiences
as a Greek mercenary in the ancient Persian empire. This work served Alexander the Great in the
preparation for his campaign into Persia.

189
190 14 Diabetes mellitus

14.1 The role of insulin and the causation of diabetes


The blood glucose concentration is normally very tightly regulated, most no-
tably by the two antagonistic hormones insulin and glucagon, such that it is
kept between 5 and 9 mM. Insulin is responsible for imposing the upper limit
on the blood glucose concentration, and the lack of insulin secretion or of in-
sulin effect are the foremost causes of diabetes. There are two major forms of
diabetes mellitus:
1. Type I is caused by a complete defect of insulin secretion, due to destruc-
tion of the β-cells in the pancreatic islets.
2. Type II is caused by a relative insensitivity of the target cells of insulin to
the effects of the hormone.
Therefore, to understand diabetes, it is crucial to understand the physiolog-
ical effects of insulin. As noted before, insulin is secreted in times of ample
glucose supply and promotes glucose utilization. Its major metabolic effects are
(1) stimulation of glycolysis and inhibition of gluconeogenesis, (2) stimulation
of glycogen synthesis and inhibition of glycogen breakdown, and (3) asimula-
tion of triacylglycerol synthesis and inhibition of its breakdown.
Insulin also controls the uptake of glucose into the cells of many tissues.
There are different types of glucose transporters in cell membranes. The main
distinctions are:
1. Sodium co-transport versus facilitated diffusion (Figure 1.7). Sodium
co-transporters are found where glucose needs to be transported against a
concentration gradient, as is the case in the intestines and the kidney tubules
(see below). This transport is always active and never regulated by insulin.
2. Insulin-dependent versus insulin-independent. This disctinction only
applies to facilitated diffusion. In insulin-dependent tissues, the hormone con-
trols the distribution of glucose transporters between the cytoplasmic mem-
brane and an inactive intracellular compartment, as discussed previously (Fig-
ure 13.5c and d).
The uptake of glucose is independent of insulin in (1) liver, (2) brain, (3) blood
cells, (4) lens and cornea of the eye. On the other hand, Insulin is required for
the uptake of glucose in muscle, fat and most other tissues. Therefore, since
without insulin most cells simply cannot import glucose, it piles up in the blood
of diabetics to very high concentrations. The high blood glucose concentration
is directly responsible for some of the symptoms of diabetes (see below).

14.2 Effects of insulin deficiency on carbohydrate


metabolism
Inhibition of glucose consumption in the peripheral tissues is only one effect
contributing to elevated blood glucose levels; equally important is the increase
of glucose formation in the liver.
14.2 Effects of insulin deficiency on carbohydrate metabolism 191

a) Glucagon Epinephrine Insulin

GR β-AR IR
AC
Phosphodiesterase 3

ATP cAMP AMP

Protein kinase A PFK-2 / Fructose-2,6-bisp’ase

Fructose-2,6-bis-P
Pyruvate dehydrogenase
PFK-1 Fructose-1,6-bisp’ase

Acetyl-CoA Pyruvate Glucose

b) Glucagon Epinephrine Insulin

GR β-AR IR
AC
Phosphodiesterase 3

ATP cAMP AMP P-Glycogen


synthase kinase
Protein kinase A Glycogen synthase
kinase
Phosphorylase kinase

Phosphorylase Glycogen synthase P-Glycogen synthase

Glycogen Glucose Glycogen

c) Insulin Glucose To liver (urea cycle / gluconeogenesis)

IR

Glucose Glutamine Alanine


GluT4

ATP α-KG Glutamate

Proteins Amino acids α-Keto-acids Pyruvate

Acetyl-CoA, ATP

Figure 14.1 Derailment of glucose and amino acid metabolism in diabetes. a: Glycol-
ysis and gluconeogenesis; b: Glycogen metabolism; c: Protein degradation in muscle.
See text for details.
192 14 Diabetes mellitus

When insulin is lacking, the insulin-antagonistic hormones glucagon and


epinephrine dominate metabolic regulation. This will drive up gluconeogenesis
and inhibit glycolysis, which effects are mediated by the intracellular second
messengers cAMP and fructose-2,6-bisphosphate (Figure 14.1a ; also see Figure
7.4). Similarly, glycogen synthesis will be inhibited and glycogen degradation
will be accelerated, by way of the previously discussed intracellular cascades
that result in the phosphorylation of both glycogen synthase, which is inac-
tivated, and phosphorylase, which is activated (Figures 14.1b and 8.5). The
rising level of blood glucose is further compounded by the derailment of mus-
cle metabolism (Figure 14.1c). Since muscle can no longer take up glucose, it
turns to protein breakdown to fill its needs for ATP. Disposal of nitrogen from
degraded amino acids generates glutamine and alanine, which are transported
to the liver, where they feed the urea cycle and gluconeogenesis as discussed
previously (Figure 12.8).

14.3 Insulin deficiency and lipid metabolism


In fat tissue, lack of insulin leads to disinhibition of hormone-sensitive lipase
(Figure 14.2). This induces breakdown of triacylglyerol to glycerol and fatty
acids, which are released into the circulation. Glycerol will be fed into gluco-
neogenesis in the liver. Fatty acids may also enter the liver and be degraded to
acetyl-CoA. The amount of acetyl-CoA in the liver is further increased by the
excess of blood glucose, which increases the intracellular glucose level (remem-
ber that the liver can take up glucose without insulin). This will drive up the
synthesis of ketone bodies, triacylglycerol and cholesterol, the levels of all of
which are increased in the blood of diabetic patients. The increased blood fat
increases the incidence and severity of atherosclerosis among diabetic patients.

14.4 Laboratory findings and clinical symptoms in acute


type I diabetes
From the foregoing, we can understand the following laboratory findings in
acute type I diabetes:
1. Increased blood glucose. The normal concentration range is 5-9 mM; in
acute diabetes, it can be several times higher.
2. Acidic deviation of the blood pH. This is mainly due to the increased
formation of ketone bodies, which are acids. This condition is called
ketoacidosis.
3. Increased blood fats (lipoproteins).
4. Increased plasma and urine levels of urea, due to the accelerated protein
breakdown in muscle.
5. Decreased levels of insulin and of C-peptide.
14.4 Laboratory findings and clinical symptoms in acute type I diabetes 193

a) Epinephrine Insulin

β-AR IR
AC

Phosphodiesterase 3

ATP cAMP AMP

Protein kinase A

Hormone-sensitive lipase

Triacylglycerol Fatty acids + glycerol

b) Glucose Fatty acids from fat tissue

Glucose Pyruvate

Acetyl-CoA Fatty acids, TAG

Ketone bodies Cholesterol Lipoproteins

Figure 14.2 Derailment of fat metabolism in diabetes. a: Disinhibition of hormone-


sensitive lipase in fat tissue triggers breakdown of triacylglycerol. b: Increased levels
of fatty acids and of glucose lead to increased formation of ketone bodies, cholesterol,
and triacylglycerol.

Clinically, acute type I diabetes is characterized by these symptoms:


1. Thirst, increased urine flow. Thirst, obviously, is due to the loss of fluid,
and this in turn is due to the osmotic activity of glucose in the urine. The
reason why diabetics lose glucose in the urine is discussed below.
2. Acetone smell. As discussed in section 10.4, acetone forms as a byprod-
uct of ketone bodies.
3. Recent loss of weight. Both protein and fat are being degraded, and the
loss of glucose in the urine can amount to a significant loss of calories.
4. In severe cases, the patients may be unconscious (comatose). Two effects
may contribute: The pH deviation (ketoacidosis), and blood plasma hyperos-
molarity, a consequence of the high glucose concentration. The high osmotic
194 14 Diabetes mellitus

activity of the blood and interstitial fluids drains water from the cells, which
hampers cell function.
5. Some patients report a recent flu-like infection, sometimes with chest
pain or even myocarditis (inflammation of the heart muscle).

14.5 The cause of β-cell destruction in type I diabetes


The last symptom in the preceding list is related to the Coxsackie virus infec-
tion that results in the destruction of the pancreatic islet β-cells. Coxsackie
viruses are small RNA viruses taxonomically related to poliovirus and hepatitis
A virus. Only certain Coxsackie virus types are prone to cause diabetes. The
mechanism of cell damage consists in an immunological cross-reaction. The im-
mune system copes with viral infections by way of destroying the virus-infected
cells. In the case of Coxsackie virus type B4, and possibly some other types,
immune cells evoked by the virus mistake surface antigens on β-cells for viral
antigens and accordingly destroy them.
The risk of a a person to suffer from diabetes as a consequence of a Cox-
sackie type B4 infection is strongly dependent on their genetic background,
more specifically their HLA genotype. For example, people with the HLA-DQ
haplotype A1: 0301-0302 / B1: 0501-0201 carry a twentyfold increased risk
to suffer diabetes, relative to the population average. Another haplotype, B1:
0602, has a relative risk of only 3% of the population average. Figure 14.3
summarizes the basics of these immunological aspects.

14.6 Why do diabetic patients lose glucose in the urine?


Glucose is precious, and in healthy individuals the concentration of glucose in
the urine is negligible. Why then does glucose appear in the urine in diabetic
patients? To understand this, let’s have a brief look at how the kidney produces
urine. The kidney contains a large number of similar functional units, each of
which is called a nephron. Within each nephron, urine production proceeds in
two stages (Figure 14.4):
1. Ultrafiltration of the blood plasma. This occurs in the glomerulus; all
small solutes found in the blood plasma (glucose, ions, amino acids and so on)
will appear in the filtrate.
2. Selective, active reuptake of solutes and of water in the tubuli of the
nephron.
The capacity of glucose reuptake is limited by the abundance of glucose
transporter molecules in the tubuli. While quite a few processes in metabolism
have a functional reserve, the transport of glucose is not one of them; its
capacity saturates just slightly above the physiological range of the glucose
concentration in the blood plasma and the primary urine filtrate.
14.6 Why do diabetic patients lose glucose in the urine? 195

a)

T T

b)

T cell
receptor

viral cellular
peptide peptide
HLA high-risk HLA
molecule molecule

c)

Figure 14.3 Immunological causation of diabetes type I. a: Basics of antiviral immu-


nity. Each cell presents peptides that result from intracellular protein breakdown on
its surface, where they are associated with HLA antigens. If a virus infects the cell, viral
peptides get presented as well. Specific T lymphocytes recognize the viral peptides via
their T cell receptors, and in response become activated, destroy the cell and propagate.
b: The T cell receptor recognizes not just the viral peptide but instead the entire HLA /
peptide complex. Some high-risk HLA antigens, when associated with innocent cellular
peptides, may mimic the structure of the viral peptide and thus induce destruction of
a non-infected cell. c, left: Structure of a HLA antigen with a peptide bound. Right: the
approximate outline of the binding site of the T cell receptor is indicated.
196 14 Diabetes mellitus

a) Blood perfusion (1500 l / day)

Filtration (150 l / day)

Reuptake of glucose and of Collection and


other solutes by specific excretion
transport, of water by osmosis (1.5 l/ day)

b) Reabsorption maximum (10 mM)


Amount
filtrated

Amount
excreted

Plasma glucose concentration

Figure 14.4 Glucose secretion in the urine in diabetes. a: The nephron and its
function. Blood plasma is filtrated in the glomerulus (top), and the filtrate passed
through the tubuli before excretion. Most of the water and many solutes, including
glucose and amino acids, are reclaimed by active transport in the tubuli. b: The
maximum rate of glucose reuptake is limited by the number of transporter molecules.
It is saturated at approximately 10 mM glucose.

14.7 Treatment of type I diabetes

The fundamental thing, of course, is to replace the lacking insulin. An acutely ill,
possibly comatose patient will require additional initial measures. Intravenous
infusion therapy is performed, with the following goals:
1. Replacement of fluid,
2. Adjustment of blood pH and electrolytes (potassium, sodium, calcium),
which are commonly derailed by the disturbed kidney function,
3. Rapid adjustment of insulin dosage, guided by frequent monitoring of
14.8 Long-term complications of diabetes mellitus 197

blood glucose.

14.8 Long-term complications of diabetes mellitus


When the acute situation has been stabilized, intravenous therapy can be discon-
tinued, and insulin can be applied by subcutaneous injection. It is important to
adjust the diet of the patient and the insulin therapy so that the blood glucose
remains as close to physiological concentrations as possible. This is necessary
to prevent or postpone the long-term complications of diabetes, which will be
caused if blood glucose is chronically elevated:2
1. Increased formation of sorbitol from glucose via the sorbitol pathway
(Figure 4.8) in the lens of the eye will induce cataract.
2. Increased conversion of glucose to fat will lead to enhanced blood fats
and promote atherosclerosis.
3. Tissue damage to various organs (peripheral nerves, kidney). The mech-
anism is not completely understood but seems to involve direct glucosylation
of proteins, and possibly again the sorbitol pathway.

14.8.1 Insulin preparations for substitution therapy

In order to keep blood glucose in a physiological range, it is necessary to


maintain the blood level of insulin be in its physiological range, too. This is
not trivial. Circulating insulin is inactivated by circulating peptidases and has a
half life of only about 25 minutes. Thus, after intravenous injection of a single
dose of insulin, the concentration declines rapidly. In contrast, the pancreas
supplies insulin continuously, with peaks after meals and a lower but fairly
steady level during the intervals between them.
In acute therapy and in a hospital setting, the insulin level can be adjusted
as needed through continuous intravenous infusion. In outpatients and for
sustained therapy, however, we need other methods to mimic or at least ap-
proximate the physiological time course.
One way to achieve a more protracted time course of the plasma insulin level
is through subcutaneous application. When insulin is injected subcutaneously,
it will wind up in the interstitial space. Insulin then has to “reversely distribute”
into the circulation. This uptake is fairly rapid for monomeric insulin, which
is small enough to diffuse through the pores in the capillary wall. However, at
high concentrations, insulin self-associates into hexamers (Figure 14.5), which
are too large for rapid capillary uptake. Only the monomers that remain present
at equilibrium will enter the bloodstream. If the injection is applied shortly
2 It is interesting to note that the tissues most severely affected by diabetic long-term complica-

tions do not require insulin to take up glucose. In these tissues, the intracellular level of glucose
will be higher than in the insulin-dependent ones.
198 14 Diabetes mellitus

before a meal, the time profile of insulin becoming available in the circulation
approximates the duration of the physiological postprandial peak.
The problem that remains then is to keep the level of insulin sufficiently
high in the periods between peaks. To this end, the rate of capillary uptake
must be slowed down even further. Preparations that have been conditioned for
delayed uptake are referred to as long-acting or “basal” insulins, as opposed to
native insulin and other short-acting “bolus” insulins. A combination of basal
and bolus insulins, and optionally intermediate-acting ones, can then be used
to mimic the overall physiological insulin secretion profile. The rate of insulin
monomer release is influenced by a multitude of effects, which can be exploited
in the preparation of long- and intermediate-acting insulins:
1. Insulin is negatively charged at neutral pH, which will cause electrostatic
mutual repulsion of the monomers, promoting dissociation. Adjusting the pH
to lower values will reduce repulsion and promote aggregation.
2. Positively charged additives such as zinc and, more strongly, protamine—
a small, positively charged nuclear protein obtained from the testes of rainbow
trout—will associate with insulin and promote its aggregation.
3. Insulin crystals dissolve more slowly than amorphous aggregates.
4. Addition of two positively charged arginine residues to the carboxy-ter-
minus of the B chain, in combination with substitution of residue asparagine
A21 by glycine, yields “glargine”, a derivative with stable, slow release kinetics.
5. Covalent linkage of a fatty acyl residue to lysine 29 yields “insulin de-
temir”, which may form micellar aggregates or bind to other proteins, causing
slower uptake into and slower clearance from the blood.
The exact mixture and dosage of slow- and fast-acting insulins has to be
adjusted empirically with each patient. Traditionally, when developing an indi-
vidual treatment plan, emphasis was placed on minimizing the number of daily
insulin injections. One limitation of this approach is that the patient needs to
carefully synchronize his meals with his insulin application schedule. Moreover,
the blood glucose level will often not be as tightly controlled as is desirable in
order to minimize the induction of diabetic long-term complications.
Tighter glucose control is the purpose of intensive insulin therapy, in which
frequent measurements of blood glucose are used to guide the likewise more
frequent applications of insulin. One risk inherent in this approach is that
hypoglycemia may result when a dose of insulin is applied before the previous
ones have been fully taken up into the circulation. In order to minimize this
risk, it is desirable to accelerate the capillary uptake beyond the rate achievable
with native insulin. Several mutant insulins have been created that aggregate
less readily than wild type insulin and therefore undergo faster capillary up-
take. Insuline lispro, in which amino acid residues proline B28 and lysine B29
are switched, and insulin aspart, which contains a mutant aspartate residue
at position B28, are in clinical use and reportedly offer a reduced risk of hy-
14.9 Glucose assays 199

Hexamer Dimer Monomer

Capillary wall

Figure 14.5 Aggregation and capillary wall penetration of insulin. Zinc ions and high
insulin concentration promote the formation of hexamers, which don’t permeate across
the capillary walls. The aggregation equilibrium can be shifted either way by various
point mutations, which is exploited in the preparation of both fast- and slow-acting
insulins (see text for details).

poglycemia. Figure 14.6 illustrates how these changes reduce the stability of
insulin aggregates.
The idea of just-in-time application of insulin leads logically to insulin
pumps, which can release insulin continuously, much like the pancreatic islets.
Ideally, the flow rate would be automatically controlled without any required
user intervention by continuous measurement of the blood glucose level. To
avoid undulations in the feedback loop, the delay between the subcutaneous re-
lease by the pump and the availability of insulin in the circulation should be as
small as possible; therefore, insulin preparations with minimized aggregation
will again be preferable.
Other insulin delivery methods have also been developed. Inhalable insulin
was available on the market for a short while. However, due to the concerns
about the long term effects of insulin on lungs and the accuracy in the dosage,
demand was lower than expected, and the product was terminated.

14.9 Glucose assays


The dosage of insulin applied has to be adjusted according to the prevailing
glucose concentration. This can be measured with a variety of enzymatic assays.
One of these is the glucose oxidase/peroxidase assay:

glucose + O2 ------→
- gluconate + H2 O2 glucose oxidase
H2 O2 + colorless pre-dye ------→
- H2 O + dye peroxidase

In this coupled assay, the amount of dye formed in the second step is propor-
tional to the glucose consumed in the first step. (Note that glucose oxidase is
isolated from microbes but does not have a role in human metabolism).
200 14 Diabetes mellitus

Insulin dimer 1

Insulin dimer 3

Insulin dimer 2

Val 3 Pro 28

Glu 21
Val 12

Tyr 16

Figure 14.6 Structure of the insulin hexamer. The hexamer is composed of three
dimers and stabilized by two centrally placed zinc ions. The two monomers of each
dimer are highlighted in dark and light shades, respectively. In each dimer, the proline
residue at position B29 of one monomer interacts with a patch of hydrophobic residues
(valine B3, valine B12 and tyrosine B16) of the other monomer. In insulin lispro, proline
B28 is replaced with lysine, which destabilizes the interaction of the two monomers. In
insuline aspart, aspartic acid replaces proline B28, which also breaks the hydrophobic
interaction and additionally creates electrostatic repulsion with glutamate B21 of the
other monomer.

As a long-term parameter of diabetes adjustment, a parameter known as


HbA1c is commonly used. HbA1 is one of the chains of hemoglobin. Its amino
terminus may undergo spontaneous, non-enzymatic reaction with glucose,
14.10 Diabetes type II 201

forming a Schiff base, which is called HbA1c :

Glucose−CH− - Glucose−CH−
−O + H2 N−hemoglobin ------→ −N−hemoglobin + H2 O

Since hemoglobin has a long lifetime, the percentage of HbA1 converted to


HbA1c reflects the average concentration of glucose over a time span of several
weeks.
As stated above, spontaneous, non-enzymatic glucosylation of proteins is
also believed to contribute to the causation of long-term complications of di-
abetes. Another potential mechanism is the lack of C-peptide. This molecule
used to be regarded only as a byproduct of insulin synthesis (Figure 13.2), and
it is not present in pharmaceutical preparations of insulin. It has been reported,
however, that treatment of diabetics with both insuline and C-peptid may re-
duce the severity of diabetic complications such as degeneration of kidneys and
peripheral nerves. Additional data from cell biological and animal experiments
support the idea that C-peptide is a mediator in its own right. No receptor for
C-peptide has been identified, however, and the effects in both experimental
animals and in patients are moderate at best. It remains to be seen whether
and when C-peptide will become part of long-term therapy in type I diabetic
patients.

14.10 Diabetes type II


In diabetes type II, there is no destruction of the pancreatic islet cells, and the
secretion of insulin is not necessarily reduced but may be in the normal range
or even enhanced. However, the peripheral tissues are less sensitive to insulin,
and so increased amounts of insulin are required to achieve the necessary effect
on the peripheral cells. The pathogenesis of diabetes type II is still not clearly
understood, so we will skip this scientific question and only note that diabetes
type II is typically associated with overweight, and is most common in elderly
patients. The intracellular metabolic dysregulation is similar to that in diabetes
type I, but typically less acute, since the residual insulin retains at least partial
effectiveness. Treatment of diabetes type II involves (1) diet to reduce weight;
often sufficient in relatively young patients, but in the long run typically not;
(2) sulfonylurea drugs such as tolbutamide, which will increase the endogenous
insulin secretion (Figure 13.3), (3) insulin therapy, just as in diabetes type I.

14.11 Diabetic coma and comatose diabetics


Untreated type I diabetes may first become manifest as diabetic coma, as stated
above; this condition is not common with untreated type II diabetes. However,
with both forms, coma may arise from an overdose of insulin, which will lead
to hypoglycemia, that is a lack of glucose in the blood. Since the brain depends
202 14 Diabetes mellitus

on glucose, it will pass out. Now, if you find a known diabetic in a comatose
state, what do you do – give him glucose or insulin? Since the hypoglycemic
coma is more immediately life-threatening than the hyperglycemic one, you
must always use glucose first. Only if that doesn’t help should you try insulin.3

14.12 Other forms of diabetes


While type I and II diabetes are the most common forms, diabetes may also
take the following forms:
1. Secondary diabetes may occur due to other endocrine diseases. Exam-
ples are diabetes due to cortisone- or epinephrine-producing tumors of the
adrenal glands; both epinephrine and cortisone have metabolic effects that are
antagonistic to insulin. Treatment consists, if possible, in curing the underlying
primary disease.
2. Drug-induced diabetes. This is most commonly observed with cortisone
and other corticosteroids, which are used to treat chronical inflammatory or
autoimmune diseases that are refractory to milder treatment. Increased blood
glucose is just one of their side effects.

14.13 The End


This concludes our overview of metabolism. If you made it this far, I hope
that you found at least some of it interesting. Please let me know about any
necessary corrections or desirable improvements to these notes. Thank you,
and, if you are student in my class, good luck for your exam.

3 This scenario is probably quite hypothetical nowadays, since fast and accurate glucose meters

are now widely available. However, it used to be much more realistic in the days when glucose
assays could only be done in the lab.

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