Metabolic Coordination Metabolic Control Signal Transduction

Metabolic interactions among the major fuel-metabolizing organs

NOTE: the brain and heart have no fuel stores The brain relies on glucose (and ketone bodies during starvation) The heart prefers fatty acids as its main fuel

Aspects of the control of blood glucose levels by pancreatic secretion of insulin and glucagon

Insulin promotes glucose use, whereas glucagon and epinephrine increase blood glucose

Actions of glucagon in liver that lead to a rise in blood glucose

NOTE the action of cAMP through protein kinase(s)

Major events in the storage, retrieval and use of fuels in the fed and unfed states and in early starvation

Purple indicates fuels imported into the tissue; green indicates fuels exported from the tissue

The metabolic abnormalities in diabetes.

The insulin deficiency blocks the uptake of glucose into muscle and adipose tissue and reduces catabolism in all tissues. In the liver, gluconeogensis from amino acids and citric acid intermediates is stimulated as cells attempt to remedy the perceived lack of usable glucose, and fatty acid oxidation and ketogenesis are also increased.

The major human endocrine glands and their central nervous system control centres

Hormone action can influence: enzyme activity (via second messengers), synthesis of specific proteins, or membrane permeability to ions or small molecules

Eukaryotic signal transduction systems involving membrane receptors (1-5) and /or second messengers (1-4)

Note: the end result of each pathway is phosphorylation of one or more proteins

Hierarchical nature of hormone action in vertebrates

Note the role of the hypothalamus in controlling the pituitary except for epinephrine

An example of feedback regulation of a hormone

The production of a peptide hormone almost always involves proteolytic cleavage of a protein precursor (The pro-hormone is cleaved differently in
different tissues to yield distinct assemblages of hormones and neurotransmitters)

Hormonal regulation by peptide hormones using the signal transduction pathway involving adenylate cyclase

Note the receptor, the transducer, the effector and the second messenger

Note the effect of caffeine

The cycle of G protein dissociation and reassociation

Activation of G proteins involves the displacement of GDP bound to the alpha subunit and dissociation of the complex from the beta and gamma.
The GTP bound state of the alpha subunit binds to adenylate cyclase and activates it, producing cAMP - the second messenger Hormone action is limited by the hydrolysis of GTP to GDP

Signal transduction pathways can also involve phosphoinositide turnover

Note the release of Ca2+ stimulates both protein kinase C and calmodulin kinase

Steroid hormones act intra-cellularly. This gives a long-term effect and usually involves the activation of specific genes

Review
Key slides

Metabolism: overview

1:1

oxidation reactions generally release energy

most living cells obtain the energy needed for cellular work by oxidizing metabolic fuels the energy yielding (catabolic) pathways of metabolism are oxidative reaction sequences that result in the transfer of electrons from fuel molecules

through a series of electron carriers and finally to oxygen

1:12

Standard Free-Energy Changes for Coupled Reactions are Additive

Go = + 10 kJ/mol Go = - 30 kJ/mol

Go = - 20 kJ/mol

Equilibrium for the overall process now lies far to the right; The consequence is that B is efficiently produced from A NOTE: This coupling of endergonic reactions to exergonic reactions is a fundamental principle in biochemistry, and is used not only to drive reactions but also for membrane transport, neuronal transmission and muscle contraction

One of the most common methods cells use to couple reactions is through the use of phosphate containing compounds
2:14

Note the range of potential transfer energy of these compounds (-60 to 10 Go kJ/mol ) This indicates that some of these phosphate hydrolysis reactions are very high energy processes while others are not.

Q: WHY do some compounds have a higher free energy of hydrolysis? Its not the phosphate bonds themselves, but rather some property of both the reactants and products in the reactions that contribute to the differences in free energy
2:17

KM practical example
Hypersensitivity to alcohol: facial flushing and tachycardia after ingesting even a small amount of ethanol
alcohol dehydrogenase aldehyde dehydrogenase

ethanol

acetaldehyde

acetate

2 isoforms #1) low KM (high affinity) mitochondrial form (not much substrate needed to activate the enzyme)

#2) high KM (low affinity) cytosolic form (lots of substrate needed to activate the enzyme
what would be the consequences of an impaired high affinity isoform of aldehyde dehydrogenase?

Acetaldehyde builds up and escapes into the bloodstream (causing the physiological effects)
Note: the mitochondrial enzyme is less active due to the substitution of a single amino acid
4:1

Competitive Inhibition continued The overall effect of competitive inhibition is that the enzyme cannot bind substrate as well when the inhibitor is present so the enzyme acts as if its KM were increased (Vmax doesnt change)

Competitive inhibition can be relieved by increasing the substrate concentration (the substrate out competes the inhibitor)
4:16

2) Non-competitive or mixed inhibitors bind at a site distinct from the substrate active site on the enzyme, but will bind to either E or ES

The overall effect is an apparent decrease in Vmax because essentially the inhibitor lowers the concentration of functional enzyme (while KM remains unaffected)

4:17

Coenzymes:
molecules bound to enzymes to help carry out the reaction like enzymes, they are either unmodified or regenerated during catalysis
coenzymes may also act as carriers of specific functional groups each kind of coenzyme has a particular chemical function cofactors can be considered a subset of coenzymes (generally metal ions) Vitamins as coenzymes: vitamins are organic molecules that are essential to the biological processes of higher organisms but cannot be synthesized by these organisms THEREFORE: vitamins must be obtained from the diet

5:1

Vitamin B12: cobalamin

vitamin B12 is not synthesized by animals or plants, only a few species of bacteria carnivores acquire B12 from meat in their diet herbivores usually depend on intestinal bacteria to synthesize B12 for them
B12 is converted in the body into two coenzymes 5-deoxyadenosylcobalamin methylcobalamin

only two actual B12 requiring reactions are known to occur in humans: conversion of methylmalonyl CoA to succinyl CoA formation of methionine from homocysteine

Regulation of enzyme activity

substrate-level control
this is a simple regulation through direct interaction of the substrates and products of each enzyme-catalyzed reaction with the enzyme itself remember: the higher the substrate concentration the more rapidly a reaction occurs until saturation of the enzyme is approached conversely: a high level of product, which can also bind to the enzyme, can inhibit the conversion of substrate to product

product can act as a competitive inhibitor

example: the first step in glycolysis is the phosphorylation of glucose to yield glucose-6phosphate glucose + ATP hexokinase glucose-6-phosphate + ADP

the enzyme hexokinase, which catalyzes the reaction, is inhibited by its product, glucose-6-phosphate THEREFORE: if glycolysis is blocked for any reason, G6P will accumulate and this accumulation will inhibit hexokinase and slow down further entry of glucose into the pathway HOWEVER: substrate level control is not sufficient for the regulation of many metabolic pathways and it is essential to have an enzyme regulated by some substance different from the substrate or immediate product
5:14

Feedback control
A
enzyme 1

enzyme 2

enzyme 3

D enzyme 4

as E accumulates it sends a signal back that inhibits A to B this is an example of negative feedback control because an increase in the concentration of E leads to a decrease in the rate of production of E

What if A feeds into 2 pathways?

A B C

There are lots of possibilities to control the pathway to keep G and N in balance G might inhibit the C to D enzyme and/or activate the C to K enzyme N might inhibit the C to K enzyme and/or activate the C to D enzyme both G and N might inhibit the A to B enzyme
5:15

Allosteric enzymes
Both inhibition and activation of enzymes are essential to regulate metabolism
Control of pathways by their end products means that the necessary inhibitions and activations must be produced by molecules that come from far down the assembly line and therefore bear no resemblance to either the substrates or the direct products of the enzymes to be regulated organisms have developed a special class of enzymes capable of allosteric regulation (where structures of regulators do not need to resemble substrate or direct product)

5:16

Covalent modification as a means of regulating enzyme activity

Enzymes can be inactive until they are activated by a covalent modification Enzymes can be active until they are inactivated by a covalent modification Some covalent modifications can be reversed, some cannot be reversed

5:19

Epimers
Two sugars that differ only in the configuration around one carbon atom are called epimers

D-glucose and D-mannose are diasterioisomers (they are not mirror images) but they differ in stereochemistry only around C-2 so they are a subset of diastereoisomers

D-glucose and D-galactose differ in stereochemistry only around C-4 Are D-mannose and Dgalactose epimers of each other?

6:11

Phosphate esters

Phosphate esters of the monosaccharides are major participants in many metabolic pathways
Condensation of phosphoric acid with one of the hydroxyl groups of a sugar forms a phosphate ester Note: the standard free energy of hydrolysis for these phosphate esters of monosaccarides are all less negative than the free energy of hydrolysis of ATP; therefore, ATP can act as a phosphate donor to monosaccharides But, since hydrolysis of the phosphate esters of sugars is thermodynamically favorable, these derivatives act as activated compounds in many metabolic reactions
6:19

Glycogen synthase reaction

1. glucose is brought into cells by the membranebound glucose transporter protein and is immediately phosphorylated by hexokinase or glucokinase to yield glucose-6-phosphate 2. glucose-6-phosphate is converted to glucose-1-phosphate by phosphoglucomutase

3. UDP-glucose is synthesized by UDP-glucose pyrophosphorylase (the reaction is drawn to the right by enzymatic cleavage of pyrophosphate to orthophosphate (catalyzed by pyrophosphatase)
4. UDP-glucose donates the glucose to a nonreducing sugar hydroxyl group

(figure 16.8)

the glycogen synthase reaction can only occur on a 4 to 8 glucose residue primer chain assembled by a protein called glycogenin
7:16

Glycogen breakdown requires three enzymes

1. glycogen phosphorylase (phosphorylase) catalyzes glycogen phosphorolysis (bond cleavage by the substitution of a phosphate group) to yield glucose-l-phosphate (G1P)

2.

Phosphoglucomutase (PGM) converts G1P to G6P

phosphorylase acts repetitively on the non-reducing ends of glycogen (or amylopectin) branches until it reaches a point four glucose residues away from an a 1 6 branch point
It cannot go any further why?
7:20

3.

glycogen debranching enzyme

(transglycosylase or glycosyltransferase)
removes glycogen's branches (at the point where phosphorylase stopped)

There are two independent catalytic activities on the same enzyme The active sites are separate from each other

transfers an a(l

4) linked trisaccharide unit to the non-reducing end of another branch

this reaction forms a new a(l 4) linkage and makes these three glucose units available for the phosphorylase reaction (phosphorolysis) also hydrolyzes the a(l 6) linkage of the remaining glucose (note hydrolyzed - not phosphorylized) to yield a glucose

Overall: in a molecule of glycogen, about 10% of the residues (those at the branch points) are converted to glucose rather than glucose-1phosphate
7:22

Carbohydrate catabolism

8:6

What happens to the lactate produced by muscle cells?

Cori cycle

What happens to lactate produced by microorganisms?

Many microorganisms ferment glucose and other hexoses to lactate Certain lactobacilli and streptococci, for example, ferment the lactose in milk to lactic acid The dissociation of lactic acid to lactate and H+ in the fermentation mixture lowers the pH, denaturing casein and other milk proteins and causing them to precipitate Under the correct, controlled conditions, the curdling process produces cheese or yogurt, depending on the microorganism

Fig. 14.22

This is really an anabolic pathway, but because it starts with the oxidation of glucose we will consider it now.

NOTE: the glucose 6-phosphate

Human genetic disorders involving pentose phosphate pathway enzymes Glucose 6-phosphate dehydrogenase deficiency
The most common human enzymopathy affects ~400 million individuals (most are asymptomatic) Causes a drug induced hemolytic anemia (because of inadequate levels of NADPH)

NADPH, a reductant in many biosynthetic pathways, also protects cells from oxidative damage by hydrogen peroxide (H2O2) and superoxide free radicals
oxidized glutathione (GSSG) is reduced (GSH) by glutathione reductase: an NADPH-dependant enzyme

The fate of carbon in the citric acid cycle

Fig. 14.3

The citric acid cycle is the hub of intermediary metabolism Fatty acids and the carbon skeletons from amino acids are also broken down into acetyl CoA

Acetyl CoA is produced from pyruvate, BUT pyruvate cannot be formed from acetyl CoA

Fatty acids: the simplest lipid molecules

a hydrophylic carboxylate group is attached to one end of a hydrocarbon tail (carboxyl group is normally ionized under physiological conditions)

stearic acid is a saturated fatty acid Saturated fatty acid: one in which all the carbons in the tail are hydrogen bonded

oleic acid is an unsaturated fatty acid Unsaturated fatty acid: a fatty acid that contains one or more double bonds

Abbreviation system for fatty acids:

(most fatty acids have an even number of carbon atoms- because of the way they are synthesized)

example: stearic acid 18:0 1. the number before the colon gives the total number of carbons 2. the number after the colon gives the number of double bonds

example: oleic acid 18:1c9 3. the configuration of the double bond is c (cis) or t (trans) 4. the numbers after the denote the carbon atom where each double bond starts (counting from the carboxyl) example: arachidonic acid 20:4c5,8,11,14

Overview of lipoprotein transport pathways and fates

Fig. 18.7

The LDL uptake by the LDL receptor

clatherin coated pits endosomes lysosomes

in lysosomes there is hydrolysis of LDL to: amino acids (from the apoprotein) free cholesterol the LDL receptor is recycled to the membrane surface to pick up more LDL (cycle of ~10 minutes)

Fig. 18.10

What happens to the free cholesterol in the cell?

much of it goes to the endoplasmic reticulum where it is used for membrane synthesis
it can also be re-esterified for storage inside the cell

Note the regulatory effects of cholesterol on ACAT and HMG-CoA reductase The Statins are inhibitors of the HMG-CoA reductase

Regulatory effects of internalized cholesterol

suppression of endogenous cholesterol synthesis by 1. inhibition of HMG-CoA reductase suppression of transcription of HMG-CoA reductase acceleration of degradation of HMG-CoA reductase
2. activation of acyl CoA:cholesterol acyltransferase (ACAT) enzyme that synthesizes cholesterol esters from cholesterol and a long-chain acyl-CoA (activating the enzyme that promotes cholesterol storage)

3. regulation of LDL receptor by decreasing mRNA for the receptor decreased synthesis of the receptor ensures that cholesterol will not be taken into the cell in excess of the cells needs, even when extra-cellular levels are high

This explains why excessive dietary cholesterol leads directly to elevations of blood cholesterol levels
With intra-cellular levels regulated so well, extra-cellular cholesterol accumulates and has nowhere else to go

Fatty acid oxidation

1904: Franz Knoop elucidated the nature of fatty acid oxidation pathway

(that fatty acids are degraded by oxidation at the carbon)

These experiments are a landmark in biochemistry because were the first to use a synthetic label (phenyl group) to follow a metabolic pathway 1949: Eugene Kennedy and Albert Lehninger demonstrated that fatty acids are oxidized in mitochondria

Fig. 18.12

Overview of the fatty acid oxidation pathway

Fig. 18.13

Acetyl-CoA is a key intermediate between fat and carbohydrate metabolism

NOTE the role of citrate in transporting acetyl units from the mitochondria to the cytosol where fatty acid biosynthesis occurs

Fig. 18.22

There are chemical similarities between the oxidation and the synthesis of a fatty acid
Fig. 18.23

Note the use of NADPH as the reducing agent in the biosynthetic pathway NAD+ and FAD are the cofactors for dehydrogenases; NADPH is the cofactor for reductases The NADPH mainly comes from the pentose phosphate pathway

Transport of acetyl units and reducing equivalents used in fatty acid synthesis

1 2 3 4 5

citrate synthase citrate lyase malate dehydrogenase (note 2 forms) malic enzyme pyruvate carboxylase

NET result is 1 molecule of acetyl-CoA and 1 molecule of NADPH produced with 1 molecule of ATP converted to ADP and Pi and 1 NADH going to NAD+ The rest of the NADPH comes from the pentose phosphate pathway

Fig. 18.31

Stage 1) Formation of Mevalonate

first part of the pathway is identical to reactions in ketogenesis But the isozymes are in the cytosol
condensation of two molecules of acetyl-CoA to give acetoacetyl-CoA acetoacetyl-CoA condenses with a third molecule of acetyl-CoA to give -hydroxy--methylglutaryl-CoA (HMG-CoA) Note that the first two reactions are reversible and do not commit the cell to the synthesis of cholesterol (or other isoprene compounds) HMG-CoA reductase catalyzes the four-electron, NADPH-dependent reduction of HMG-CoA to mevalonate HMG-CoA reductase is an integral membrane protein of the smooth endoplasmic reticulum this is the primary regulatory step of cholesterol synthesis

Glutamate Dehydrogenase
NOTE: this reaction is reversible, but acts primarily in the direction of glutamate formation in most bacteria and many plants

Page 714

Glutamine Synthetase

Fig. 20.9

Fates of Amino Acid Carbon Skeletons

Fig. 20.12 NOTE: glucogenic and ketogenic amino acids * indicates that there is more than one route of entry

Removal of a-amino groups:

the first step in the catabolism of amino acids once they have reached the liver

Transamination reactions: the a-amino group is transferred to the a-carbon of a -ketoglutarate generating glutamate and the corresponding a -keto acid
There is no net deamination

The purpose is to collect the amino groups from any amino acid undergoing degradation in the form of L-glutamate

This glutamate then serves as an amino acid group donor for biosynthetic pathways or for the excretory pathway that leads to the elimination of nitrogenous waste products

Alanine transports ammonia from muscles to the liver glucose-alanine cycle

If you are using muscle protein for fuel and there is sufficient pyruvate, the amino group will often be added to pyruvate to form alanine Alanine enters the circulation and in the liver is transaminated back to pyruvate and the amino group is now on glutamate Glutamate enters the mitochondria for further processing by glutamate dehydrogenase and the pyruvate can be used for gluconeogenesis

Remember in the Cori cycle lactate from muscle is sent to the liver for gluconeogenesis

NOTE: the enzymes involved in these reactions

See pages 643-645 in Berg et al. This textbook does not describe this at all well!

Fig. 20.14

Krebs-Henseleit Urea Cycle

Fig. 20.13

NOTE: some parts are cytosolic and others occur in the mitochondria

Urea is produced from ammonia in five enzymatic steps

Step 1: the formation of carbamoyl phosphate occurs in the mitochondrial matrix

However the ammonia is generated, it is immediately used in a reaction with CO2 (in the form of bicarbonate) produced by mitochondrial respiration to form carbamoyl phosphate The ATP dependent reaction is catalyzed by carbamoyl phosphate synthetase I Note: carbamoyl phosphate synthetase II is a cytosolic enzyme involved in pyrimidine biosynthesis (see later) The carbamoyl phosphate can be considered an activated compound and now enters the urea cycle

The big picture

Transport across the mitochondrial membrane

Histidine can be decarboxylated to histamine

Histamine is a vasodilator in animal tissue and is released in large amounts as part of the allergic reaction
Histamine also stimulates acid secretion in the stomach by binding to a histamine receptor Many drugs interfere with either the synthesis of histamine or the action of histamine

Cimetidine is a histamine receptor antagonist and structural analog of histamine that promotes healing of duodenal ulcers by inhibiting secretion of gastric acid

De novo synthesis of purine nucleotides

De Novo Purine Nucleotide Synthesis Begins with PRPP

In the first committed step of the pathway: Glutamine donates an amino group to C-1 of PRPP

5-phosphoribosylamine: is very unstable Half-life of 30s at pH 7.5

The purine ring is built on 5-phosphoribosylamine

Conversion of inosinate to adenylate (AMP) requires the addition of an amino group from aspartate in a two step reaction using GTP

Conversion of inosinate to guanylate (GMP) requires an NAD+ dependent oxidation of inosinate at C-2 followed by the addition of an amine group from glutamate in a reaction using ATP

Step 1: The carbamoyl phosphate in pyrimidine biosynthesis is made in the cytosol by carbamoyl phosphate synthetase II

Step 2: carbamoyl phosphate reacts with aspartate to produce carbamoyl aspartate (committed step) Enzyme is aspartate transcarbamoylase Step 3: dehydration and ring closure to form dihydroorotate Step 4: Oxidation to orotate In mammals, the first three enzymes are part of a large multienzyme complex (CAD) Step 5: Once orotate is formed, PRPP donates a ribose-5 phosphate side chain Step 6: decarboxylation to uridine monophosphate (UMP) Step 7 and 8: UMP is phosphorylated to UTP By cytidylate synthetase to CTP

The nitrogen donor is usually glutamine

Thymidylate synthase reaction

Conversion of dUMP to dTMP

1: a one carbon unit from N5, N10 methylene tetrahydrofolate is transferred to dUMP To make dTMP

2: during this reaction, the N5, N10 methylene tetrahydrofolate is oxidized to dihydrofolate

This regeneration is essential for the many processes that rely on tetrahydrofolate

3: Dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase

Thymidylate synthase and folate metabolism as chemotheraputic targets

Methotrexate is a competitive inhibitor of dihydrofolate reductase (binds enzyme 100 fold more tightly than dihydrofolate)

THE END THANKS AND GOOD LUCK IN THE FINAL EXAM