Biochemistry,

study of the chemical substances and processes that occur in plants, animals, and microorganisms
and of the changes they undergo during development and life.
It deals with the chemistry of life, and as such it draws on the techniques of analytical, organic,
and physical chemistry, as well as those of physiologists concerned with the molecular basis of
vital processes.
All chemical changes within the organismeither the degradation of substances, generally to
gain necessary energy, or the buildup of complex molecules necessary for life processesare
collectively termed metabolism.
These chemical changes depend on the action of organic catalysts known as enzymes, and
enzymes, in turn, depend for their existence on the genetic apparatus of the cell. It is not
surprising, therefore, that biochemistry enters into the investigation of chemical changes in
disease, drug action, and other aspects of medicine, as well as in nutrition, genetics, and
agriculture.

Overview of Metabolism
Introduction:

Metabolism is the sum total of all chemical reactions involved in maintaining the living state of
the cells, and thus the organism. In general metabolism may be divided into two categories:
catabolism or the break down of molecules to obtain energy; and anabolism or the synthesis of
all compounds needed by the cells (examples are DNA, RNA, an protein synthesis). The diagram
on the left contains a summary of all the types of metabolism that will be examined. In this
module, the electron transport chain is examined.

Bioenergetics is a term which describes the biochemical or metabolic pathways by which the cell
ultimately obtains energy.

Nutrition is a science that deals with the relation of food substance to living things. In the study
of nutrition, the following items must be considered: a) bodily requirement for various
substances; b) function in body; c) amount needed; d) level below which poor health results.
Essential foods supply energy (calories) and supply the necessary chemicals which the body
itself cannot synthesize. Food provides a variety of substances that are essential for the building,
upkeep, and repair of body tissues, and for the efficient functioning of the body.

A complete diet must supply the elements; carbon, hydrogen, oxygen, nitrogen, phosphorus,
sulfur, and at least 18 other inorganic elements. The major elements are supplied in
carbohydrates, lipids, and protein. In addition, at least 17 vitamins and water are necessary. If an
essential nutrient is omitted from the diet, certain deficiency symptoms appear.

Minerals:

The minerals in foods do not contribute directly to energy needs but are important as body
regulators and as essential constituents in many vital substances within the body. A MINERAL is
rather loosely defined as any element not normally a part of the structures of carbohydrates,
proteins, and fats. More than 50 elements are found in the human body.

About 25 elements have been found to be essential, since a deficiency produces specific
deficiency symptoms. All of the minerals required by the human body are probably not known at
this time. Although minerals may not be part of the structures of carbohydrates, proteins, and
fats, they are mixed in the foods in trace amounts during the growing process by uptake from the
soil.

Major Minerals Include: calcium, phosphorus, iron, sodium, potassium, and chloride ions.

Other Essential Minerals Include: copper, cobalt, manganese, zinc, magnesium, fluorine, and
iodine.

Vitamins:

Vitamins are essential organic compounds that the human body cannot synthesize by itself and
must therefore, be present in the diet. The term vitamin (vital amines) was coined by Casmir
Funk from the Latin vita meaning "life" (essential for life) and amine because he thought that all
of these compounds contained an amine functional group.

Vitamins particularly important in metabolism include:

Vitamin A: The yellow and green pigments found in vegetables are called carotenes which are
pro vitamins and are converted into Vitamin A. The role of vitamin A in Vision has already been
discussed in a previous page.

Vitamin B2 is better known as riboflavin and is widely distributed in many foods. Riboflavin is
used to form a coenzyme FAD important in the utilization of oxygen in the cells.

Niacin, also known as nicotinic acid, is also in the B complex of vitamins. Nicotinic acid was
first obtained from the alkaloid nicotine in tobacco and was later found in many plant and animal
tissues as niacin.

Nicotinamide is a part of the important coenzyme, Nicotinamide Adenine Dinucleotide (NAD).

This NAD+ coenzyme is important during biological oxidations and is discussed in detail in a
later page.

Pantothenic Acid is art of the structure of coenzyme A.

Carbohydrates:

Foods supply carbohydrates in three forms: starch, sugar, and cellulose (fiber). Starch and sugar
are major and essential sources of energy for humans. A lack of carbohydrates in the diet would
probably result in an insufficient number of calories in the diet. Cellulose furnishes bulk in the
diet.

Since the tissues of the body need glucose at all times, the diet must contain substances such as
carbohydrates or substances which will yield glucose by digestion or metabolism. For the
majority of the people in the world, more than half of the diet consists of carbohydrates from
rice, wheat, bread, potatoes, macaroni.

Proteins:

All life requires protein since it is the chief tissue builder and part of every cell in the body.
Among other functions, proteins help to: make hemoglobin in the blood that carries oxygen to
the cells; form anti-bodies that fight infection; supply nitrogen for DNA and RNA genetic
material; and supply energy.
Proteins are necessary for nutrition because they contain amino acids. Among the 20 or more
amino acids, the human body is unable to synthesize 8, therefore, these amino acids are called
essential amino acids. A food containing protein may be of poor biological value if it is deficient
in one or more of the 8 essential amino acids: lysine, tryptophan, methionine, leucine, isoleucine,
phenylalanine, valine, and threonine. Proteins of animal origin have the highest biological value
because they contain a greater amount of the essential amino acids. Foods with the best quality
protein are listed in diminishing quality order: whole eggs, milk, soybeans, meats, vegetables,
and grains.

Fats and Lipids:

Fats are concentrated sources of energy because they give twice as much energy as either
carbohydrates or protein on a weight basis. The functions of fats are to: make up part of the
structure of cells, form a protective cushion and heat insulation around vital organs, carry fat
soluble vitamins, and provide a reserve storage for energy.

Three unsaturated fatty acids which are essential include: linoleic, linolinic, and arachidonic and
have 2, 3, and 4 double bonds respectively. Saturated fats, along with cholesterol, have been
implicated in arteriosclerosis, "hardening of the arteries". For this reason, the diet should be
decreased in saturated fats (animal) and increased in unsaturated fat (vegetable).

a) MH + NAD+ ---> NADH + H+ + M + energy

b) ADP + P + energy ---> ATP + H2O

The overall reaction for the combustion of glucose is written:

C6H12O6 + 6 O2 -----> 6 CO2 + 6 H2O + energy

Although the above equation represents the overall metabolic reaction for carbohydrates, there
are actually over thirty individual reactions. Each reaction is controlled by a different enzyme.
The failure of an enzyme to function may have serious and possibly fatal consequences. Slightly
less than half of the 686 kcal/mole of the energy produced by combustion is available for storage
and use by the cell with the remaining amount dissipated as heat.

Carbohydrates

Carbohydrates are among the most abundant compounds on earth. They are normally broken
down into five major classifications of carbohydrates:

1. Monosaccharides

2. Disaccharides

3. Oligosaccharides

4. Polysaccharides

5. Nucleotides

Monosaccharides

The word monosaccharide is derived from mono, meaning "one", and saccharide, meaning
"sugar". The common monosaccharides are glucose, fructose, and galactose. Each simple sugar
has a cyclic structure and is composed of carbon, hydrogen and oxygen in ratios of 1:2:1
respectively. Although each sugar mainly exists as a cyclic compound, it is important to note that
they are all in equilibrium to a small extent with their linear forms.

Figure %: Monosaccharides
While galactose and glucose are composed of six-membered rings, fructose has only five carbon
atoms bonded to each other in ring form.

Glucose
Glucose is the main sugar metabolized by the body for energy. The D-isomer of glucose
predominates in nature and it is for this reason that the enzymes in our body have adapted to
binding this form only. Since it is an important energy source, the concentration of glucose in the
bloodstream usually falls within a narrow range of 70 to 115mg/100 ml of blood. Sources of
glucose include starch, the major storage form of carbohydrate in plants.

Galactose

Galactose is nearly identical to glucose in structure except for one hydroxyl group on carbon
atom number four of the six-sided sugar. Since it differs in only one position about all six
asymmetric centers in the linear form of the sugar, galactose is known as an epimer of glucose.
Galactose is not normally found in nature in large quantities, however it combines with glucose
to form lactose in milk. After being absorbed by the body, galactose is converted into glucose by
the liver so that it can be used to provide energy for the body. Both galactose and glucose are
very stable in solution because they are able to adopt chair and boat conformations.

Figure %: Chair and Boat Conformations

These conformations are most stable because their OH groups are pointed away from the
structure, preventing steric hindrance.

Fructose
Fructose is a structural isomer of glucose, meaning it has the same chemical ormula but a
completely different three-dimensional structure. The main difference is that fructose is a ketone
in its linear form while glucose is an aldehyde. Through an intramolecular addition reaction with
the C-5 OH group, glucose forms a six-membered ring while fructose forms a five-membered
ring as seen in Figure 1. Upon consumption, fructose is absorbed and converted into glucose by
the liver in the same manner as lactose. Sources of fructose include fruit, honey and high-
fructose corn syrup.

Disacharides

Disaccharides, meaning "two sugars", are commonly found in nature as sucrose, lactose and
maltose. They are formed by a condensation reaction where one molecule of water condenses or
is released during the joining of two monosaccharides. The type of bond that is formed between
the two sugars is called a glycosidic bond.

Figure %: Condensation Reaction resulting in Glycosidic Bonds in Maltose

Lactose

Lactose is a disaccharide formed through the condensation of glucose and galactose. The bond
formed between the two monosaccharides is called a beta glycosidic bond (). The alpha
glycosidic bond, found in sucrose and maltose, differs from the beta glycosidic bond only in the
angle of formation between the two sugars. Unfortunately, unlike alpha glycosidic bonds, beta-
glycosidic bonds are unable to be digested by some people. Therefore, many people are lactose
intolerant and suffer from intestinal cramping and bloating due to the incomplete digestion of the
substance.

Sucrose

Sucrose is found in common table sugar and is composed of glucose and fructose linked via a 1-
2 alpha glycosidic bond.
 Figure %: Sucrose
Sucrose is an excellent preservative because it has no "reducing end" or reactive group like the
other sugars. Because glucose is joined to the carbon atom labeled number two on fructose,
neither monosaccharide is able to open or react with other compounds in solution. It is for this
reason that sucrose is an excellent natural preservative and is found in many jarred foods
including jams. Other natural sources of sucrose are found in plants such as sugar cane, sugar
beets, and maple syrup.

Maltose

Maltose is the final disaccharide and consists of two glucose molecules joined by an alpha
glycosidic bond. Maltose is an interesting compound because of its use in alcohol production.
Through a process called fermentation, glucose, maltose and other sugars are converted to
ethanol by yeast cells in the absence of oxygen. Through an analogous process, muscle cells
convert glucose into lactic acid to obtain energy while the body operates under anaerobic
conditions. Although maltose is uncommon in nature, it can be formed through the breakdown of
starch by the enzymes of the mouth.

Oligosaccharides and Polysaccharides

Carbohydrates that contain more than two simple sugars are called oligosaccharides or
polysaccharides, depending upon the length of the structure. Oligosaccharides usually have
between three and ten sugar units while polysaccharides can have more than three thousand
units. These large structures are responsible for the storage of glucose and other sugars in plants
and animals.

Oligosaccharides

Important oligosaccharides are raffinose and stachyose. Composed of repeating units of

galactose, glucose and fructose, these oligosaccharides are of nutritional importance because
they are found in beans and legumes. Because of their unique glycosidic bonds, raffinose and
stachyose cannot be broken down into their simple sugars. Therefore, they cannot be absorbed by
the small intestine and are often metabolized by bacteria in the large intestine to form unwanted
gaseous byproducts. Commercial enzyme preparations such as Beano can be consumed before a
meal rich in beans and legumes in order to aid the small intestine in the breakdown of these
oligosaccharides.
Polysaccharides

Polysaccharides or complex carbohydrates are usually monomers and consist of thousands of

repeating glucose units. Naturally, they allow for the storage of large quantities of glucose.
Starch is the major storage form of carbohydrate in plants and has two different types: amylose
and amylopectin. Although digestible alpha glycoisidic bonds link both types of starch, each type
is unique in their branching of glucose. While amylose is a straight chain polymer, amylopectin
is highly branched. These differences account for the fact that amylopectin can form stable starch
gels which are able to retain water while amylose is unable to do so. Therefore, amylopectin is
often used by manufacturers to produce many different kinds of thick sauces and gravies.
Sources of starch include potatoes, beans, bread, pasta, rice and other bread products

Like amylopectin, glycogen is a highly branched polymer of glucose that is the main storage
form of carbohydrate in humans. The main chain of the structure is composed of alpha 1, 4
glycosidic bonds, while alpha 1,6 glycosidic bonds give rise to the branch points of the polymer
(figure 5). Glycogen is stored in the liver and muscle where it is synthesized and degraded
depending upon the energy requirements of the body.

Indigestible forms of polysaccharides are known as dietary fiber and come in many different
forms including cellulose, hemicellulose, pectin, gum and mucilage. Cellulose is by far the most
abundant biochemical compound on the earth because it forms part of the structure of many
plants. It is unique among polysaccharides in that it forms intramolecular hydrogen bonds
between adjacent glucose units as well as beta 1,4 glycosidic bonds present in other
carbohydrates. These special bonding characteristics allow cellulose to form long, straight chains
of glucose and give it strength and rigidity that many plants require for proper growth. Cellulose
and most forms of hemicellulose are insoluble fibers while pectin, gum and mucilage are all
soluble fibers and readily dissolve or swell when mixed with water.

Nucleotides

Other sugars of importance are found in nucleotides such as deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). Both RNA and DNA are five sided cyclic sugars; however, RNA has
one more hydroxyl group than DNA. Glucose-6-phosphate, an intermediate in the breakdown of
glucose for energy, can be used for the synthesis of these compounds.

Carbohydrates have six major functions within the body:

1. Providing energy and regulation of blood glucose

2. Sparing the use of proteins for energy

3. Breakdown of fatty acids and preventing ketosis

4. Biological recognition processes

 5. Flavor and Sweeteners

6. Dietary fiber

Providing energy and regulating blood glucose

Glucose is the only sugar used by the body to provide energy for its tissues. Therefore, all
digestible polysaccharides, disaccharides, and monosaccharides must eventually be converted
into glucose or a metabolite of glucose by various liver enzymes. Because of its significant
importance to proper cellular function, blood glucose levels must be kept relatively constant.

Among the enormous metabolic activities the liver performs, it also includes regulating the level
of blood glucose. During periods of food consumption, pancreatic beta cells sense the rise in
blood glucose and begin to secrete the hormone insulin. Insulin binds to many cells in the body
having appropriate receptors for the peptide hormone and causes a general uptake in cellular
glucose. In the liver, insulin causes the uptake of glucose as well as the synthesis of glycogen, a
glucose storage polymer. In this way, the liver is able to remove excessive levels of blood
glucose through the action of insulin.

In contrast, the hormone glucagons is secreted into the bloodstream by pancreatic alpha cells
upon sensing falling levels of blood glucose. Upon binding to targeted cells such as skeletal
muscle and brain cells, glucagon acts to decrease the amount of glucose in the bloodstream. This
hormone inhibits the uptake of glucose by muscle and other cells and promotes the breakdown of
glycogen in the liver in order to release glucose into the blood. Glucagon also promotes
gluconeogenesis, a process involving the synthesis of glucose from amino acid precursors.
Through the effects of both glucagon and insulin, blood glucose can usually be regulated in
concentrations between 70 and 115mg/100 ml of blood.

Other hormones of importance in glucose regulation are epinephrine and cortisol. Both hormones
are secreted from the adrenal glands, however, epinephrine mimics the effects of glucagon while
cortisol mobilizes glucose during periods of emotional stress or exercise.
Despite the liver's unique ability to maintain homeostatic levels of blood glucose, it only stores
enough for a twenty-four hour period of fasting. After twenty four hours, the tissues in the body
that preferentially rely on glucose, particularly the brain and skeletal muscle, must seek an
alternative energy source. During fasting periods, when the insulin to glucagons ratio is low,
adipose tissue begins to release fatty acids into the bloodstream. Fatty acids are long
hydrocarbon chains consisting of single carboxylic acid group and are not very soluble in water.
Skeletal muscle begins to use fatty acids for energy during resting conditions; however, the brain
cannot afford the same luxury. Fatty acids are too long and bulky to cross the blood-brain barrier.
Therefore, proteins from various body tissues are broken down into amino acids and used by the
liver to produce glucose for the brain and muscle. This process is known as gluconeogenesis or
"the production of new glucose." If fasting is prolonged for more than a day, the body enters a
state called ketosis. Ketosis comes from the root word ketones and indicates a carbon atom with
two side groups bonded to an oxygen atom. Ketones are produced when there is no longer
enough oxaloacetate in the mitochondria of cells to condense with acetyl CoA formed from fatty
acids. Oxaloacetate is a four-carbon compound that begins the first reaction of the Krebs Cycle, a
cycle containing a series of reactions that produces high-energy species to eventually be used to
produce energy for the cell. Since oxaloacetate is formed from pyruvate (a metabolite of
glucose), a certain level of carbohydrate is required in order to burn fats. Otherwise, fatty acids
cannot be completely broken down and ketones will be produced
Sparing Protein and Preventing Ketosis

So why are carbohydrates important if the body can use other carbon compounds such as fatty
acids and ketones as energy? First of all, maintaining a regular intake of carbohydrates will
prevent protein from being used as an energy source. Gluconeogenesis will slow down and
amino acids will be freed for the biosyntheses of enzymes, antibodies, receptors and other
important proteins. Furthermore, an adequate amount of carbohydrates will prevent the
degradation of skeletal muscle and other tissues such as the heart, liver, and kidneys. Most
importantly, ketosis will be prevented. Although the brain will adapt to using ketones as a fuel, it
preferentially uses carbohydrates and requires a minimum level of glucose circulating in the
blood in order to function properly. Before the adaptation process occurs, lower blood glucose
levels may cause headaches in some individuals. To prevent these ketotic symptoms, it is
recommended that the average person consume at least 50 to 100g of carbohydrates per day.

Although the processes of protein degradation and ketosis can create problems of their own
during prolonged fasting, they are adaptive mechanisms during glucose shortages. In summary,
the first priority of metabolism during a prolonged fast is to provide enough glucose for the brain
and other organs that dependent upon it for energy in order to spare proteins for other cellular
functions. The next priority of the body is to shift the use of fuel from glucose to fatty acids and
ketone bodies. From then on, ketones become more and more important as a source of fuel while
fatty acids and glucose become less important.

Flavor and Sweeteners

A less important function of carbohydrates is to provide sweetness to foods. Receptors located at

the tip of the tongue bind to tiny bits of carbohydrates and send what humans perceive as a
"sweet" signal to the brain. However, different sugars vary in sweetness. For example, fructose is
almost twice as sweet as sucrose and sucrose is approximately 30% sweeter than glucose.

Sweeteners can be classified as either nutritive or alternative. Nutritive sweeteners have all been
mentioned before and include sucrose, glucose, fructose, high fructose corn syrup, and lactose.
These types of sweeteners not only impart flavor to the food, but can also be metabolized for
energy. In contrast, alternative sweeteners provide no food energy and include saccharin,
cyclamate, aspartame, and acesulfame. Controversy over saccharin and cyclamate as artificial
sweeteners still exists but aspartame and acesulfame are used extensively in many foods in the
United States. Aspartame and acesulfame are both hundreds of times sweeter than sucrose but
only acesulfame is able to be used in baked goods since it is much more stable than aspartame
when heated.

Dietary Fiber

Dietary fibers such as cellulose, hemicellulose, pectin, gum and mucilage are important
carbohydrates for several reasons. Soluble dietary fibers like pectin, gum and mucilage pass
undigested through the small intestine and are degraded into fatty acids and gases by the large
intestine. The fatty acids produced in this way can either be used as a fuel for the large intestine
or be absorbed into the bloodstream. Therefore, dietary fiber is essential for proper intestinal
health.

In general, the consumption of soluble and insoluble fiber makes the elimination of waste much
easier. Since dietary fiber is both indigestible and an attractant of water, stools become large and
soft. As a result, feces can be expelled with less pressure. However, not enough fiber
consumption will change the constitution of the stool and increase the amount of force required
during defecation. Excessive pressure during the elimination of waste can force places in the
large intestine wall out from between bands of smooth muscle to produce small pouches called
diverticula. Hemorrhoids may also result from unnecessary strain during defecation.

The disease of having many diverticula in the large intestine is known as diverticulosis. Although
diverticula is often asymptomatic, food particles become trapped in their folds and bacteria begin
to metabolize the particles into acids and gases. Eventually, the diverticula may become
inflamed, a condition known as diverticulitis. To combat the disease, antibiotics are administered
to the patient to destroy the bacteria while the intake of fiber in the diet is decreased until the
inflammation has subsided. Once the inflammation has been reduced, a high fiber diet is begun
to prevent a relapse.

Besides the prevention of intestinal disease, diets high in fiber have other health benefits. High
fiber intake reduces the risk of developing obesity by increasing the bulk of a meal without
yielding much energy. An expanded stomach leads to satisfaction despite the fact that the caloric
intake has decreased.

Beyond dieters, diabetics can also benefit from consuming a regular amount of dietary fiber.
Once in the intestine, it slows the absorption of glucose to prevent a sudden increase in blood
glucose levels. A relatively high intake of fiber will also decrease the absorption of cholesterol, a
compound that is thought to contribute to atherosclerosis or scarring of the arteries. Serum
cholesterol may be further reduced by a reduction in the release of insulin after meals. Since
insulin is known to promote cholesterol synthesis in the liver, a reduction in the absorption of
glucose after meals through the consumption of fiber can help to control serum cholesterol
levels. Furthermore, dietary fiber intake may help prevent colon cancer by diluting potential
carcinogens through increased water retention, binding carcinogens to the fiber itself and
speeding the passage of food through the intestinal tract so that cancer-causing agents have less
time to act.

Biological Recognition Processes

Carbohydrates not only serve nutritional functions, but are also thought to play important roles in
cellular recognition processes. For example, many immunoglobulins (antibodies) and peptide
hormones contain glycoprotein sequences. These sequences are composed of amino acids linked
to carbohydrates. During the course of many hours or days, the carbohydrate polymer linked to
the rest of the protein may be cleaved by circulating enzymes or be degraded spontaneously. The
liver can recognize differences in length and may internalize the protein in order to begin its own
degradation. In this way, carbohydrates may mark the passage of time for proteins.

Metabolism of Carbohydrates and Exercise

Since all digestible forms of carbohydrates are eventually transformed into glucose, it is
important to consider how glucose is able to provide energy in the form of adenosine
triphosphate (ATP) to various cells and tissues. Glucose is metabolized in three stages:

1. glycolysis

2. the Krebs Cycle

3. oxidative phosphorylation
During exercise, hormonal levels shift and this disruption of homeostasis alters the metabolism
of glucose and other energy-bearing molecules. Therefore, in this SparkNote the metabolism of
carbohydrates will be considered in the context of exercise strategies and hypotheses.

Glycolysis

The breakdown of glucose to provide energy begins with glycolysis. To begin with, glucose
enters the cytosol of the cell, or the fluid inside the cell not including cellular organelles. Next,
glucose is converted into two, three-carbon molecules of pyruvate through a series of ten
different reactions. A specific enzyme catalyzes each reaction along the way and a total of two
ATP are generated per glucose molecule. Since ADP is converted to ATP during the breakdown
of the substrate glucose, the process is known as substrate-level phosphorylation. During the
sixth reaction, glyceraldehyde 3-phosphate is oxidized to 1,3 bisphosphoglycerate while reducing
nicotinamide adenosine dinucleotide (NAD) to NADH, the reduced form of the compound.
NADH is then shuttled to the mitochondria of the cell where it is used in the electron transport
chain to generate ATP via oxidative phosphorylation, which will be described later.

The most important enzyme in glycolysis is called phosphofructokinase (PFK)and catalyzes the
third reaction in the sequence. Since this reaction is so favorable under physiologic conditions, it
is known as the "committed step" in glycolysis. In other words, glucose will be completely
degraded to pyruvate after this reaction has taken place. With this in mind, PFK seems as if it
would be an excellent site of control for glucose metabolism. In fact, this is exactly the case.
When ATP or energy is plentiful in the cell, PFK is inhibited and the breakdown of glucose for
energy slows down. Therefore, PFK can regulate the degradation of glucose to match the energy
needs of the cell. This type of regulation is a recurring theme in biochemistry.

Krebs Cycle and Oxidative Phosphorylation/Electron Transport Chain

There are many compounds that are formed and recycled during the Krebs Cycle (Citirc Acid
Cycle). These include oxidized forms of nictotinamide adenine dinucleotide (NAD+) and flavin
adenine dinucleotide (FAD) and their reduced counterparts: NADH and FADH2. NAD+ and
FAD are electron acceptors and become reduced while the substrates in the Krebs Cycle become
oxidized and surrender their electrons.
 Figure %: The Krebs Cycle

The Krebs Cycle begins when the pyruvate formed in the cytoplasm of the cell during glycolysis
is transferred to the mitochondria, where most of the energy inherent in glucose is extracted. In
the mitochondria, pyruvate is converted to acetyl CoA by the enzyme pyruvate carboxlase. In
general, Acetyl-CoA condenses with a four carbon compound called oxaloacetate to form a six
carbon acid. This six-carbon compound is degraded to a five and four carbon compound,
releasing two molecules of carbon dioxide. At the same time, two molecules of NADH are
formed. Finally, the C-4 carbon skeleton undergoes three additional reactions in which guanosine
triphosphate (GTP), FADH2 and NADH are formed, thereby regenerating oxaloacetate. FADH2
and NADH are passed on to the electron transport chain (see below) that is embedded in the
inner mitochondria membrane. GTP is a high-energy compound that is used to regenerate ATP
from ADP. Therefore, the main purpose of the Krebs Cycle is to provide high-energy electrons in
the form of FADH2 and NADH to be passed onward to the electron transport chain.

The high-energy electrons contained in NADH and FADH2 are passed on to a series of enzyme
complexes in the mitochondrial membrane.
 Figure %: Electron Transport Chain
Three complexes work in sequence to harvest the energy in NADH and FADH2 and convert it to
ATP: NADH-Q reductase, cytochrome reductase and cytochrome oxidase. The final electron
acceptor in the electron transport chain is oxygen. Each successive complex is at lower energy
than the former so that each can accept electrons and effectively oxidize the higher energy
species. In effect, each complex harvests the energy in these electrons to pump protons across the
inner mitochondria membrane, thereby creating a proton gradient. In turn, this electropotential
energy is converted to chemical energy by allowing proton flux back down its chemical gradient
and through specific proton channels that synthesize ATP from ADP. Approximately two
molecules of ATP are produced during the Kreb cycle reactions, while approximately 26 to 30
ATP are generated by the electron transport chain. In summary, the oxidation of glucose through
the reduction of NAD+ and FADH is coupled to the phosphorylation of ADP to produce ATP.
Hence, the process is known as oxidative phosphorylation.

Metabolism of Glucose and Exercise

Despite oxidative phosphorylation's large capacity for energy production, the rate of electron
transport and therefore ATP generation is limited by oxygen, the final electron acceptor/oxidizing
agent in the chain. Oxygen is readily available to the cell through circulating hemoglobin but can
only be used when the energy requirements of the cell do not exceed the rate of ATP production
via oxidative phosphorylation. This is often the case because the rate at which electrons can be
transferred to oxygen is relatively slow. In its search for a more readily available energy
production pathway, the body will switch to anaerobic glycolysis as its primary provider. Despite
requiring tendifferent reactions to form its end product pyruvate, glycolysis occurs quite rapidly.
During these ten reactions, two net ATP are formed per molecule of glucose, which help working
muscles satisfy energy requirements quickly. However, upon inspection of the Krebs Cycle
figure above, it is clear that ATP generation through this process would stop abruptly if NAD+
were not regenerated to act as an electron acceptor in the conversion of glyceraldehyde 3-
phosphate to 1,3 biphosphoglycerate. The body has adapted to this requirement by using
pyruvate to oxidize (remove electrons) from NADH, thereby forming NAD+ and lactic acid in an
oxidation-reduction reaction. An enzyme known as lactate dehydrogenase carries out this
conversion, allowing NAD+ to be recycled quickly for repeated use in glycolysis.

Like most things that seem too good to be true, there is a problem with anaerobic glycolysis. The
production of lactic acid during this process can decrease the pH of active local tissue enough to
cause cramping and fatigue. At moderate exercise intensities, the body does have a limiting
mechanism to cope with this entirely new problem. While working muscle releases lactic acid
into the bloodstream during anaerobic exercise, the liver is busy absorbing and converting lactic
acid back to pyruvate by the same enzyme found in anaerobic glycolysis. Pyruvate is then
recycled into circulation to feed working tissue. In this way, part of the metabolic burden is
shifted to the liver. If oxygen is available and aerobic conditions persist, pyruvate can enters the
mitochondria to be used in the Krebs cycle and electron transport chain if oxygen is available.
This relatively simple process is known as the Cori cycle.

Figure %: Cori Cycle

During aerobic conditions, the level of lactic acid can be maintained at a low level in the blood
for a significant period of time through the Cori cycle. The greater the fitness level of an
individual, the longer the lactic acid level can be maintained. However, even the best of active
athletes reach a point where their bodies begin to produce more lactic acid than their liver can
metabolize and it begins to accumulate in the blood. This point is known as the lactic acid
threshold for that individual and is dependent mostly on cardiac performance if aerobic
conditions persist.

Exercise strategies, weight loss and carbohydrates

The metabolism of carbohydrates can also be considered in the context of exercise strategies. For
example, what kinds of foods should be eaten before a particular type of exercise? For almost
any activity, most dieticians suggest a diet high in starch and other complex carbohydrates. Since
this is also recommended for most diets in general, eating a diet high in carbohydrates should not
be difficult. Furthermore, dieticians also recommend that people avoid meals high in fats and
proteins before exercise, since these foods inhibit gastric emptying and require longer to digest
than other foods. Since the sympathetic nervous system inhibits the gastrointestinal tract during
exercise, any remaining quantities of food in the stomach may lead to cramping and acid reflux
into the esophagus. Lastly, simple sugars that are readily absorbed by the intestine should be
avoided because they cause rapid fluctuations in blood glucose, thereby affecting the circulating
energy supply. Unlike simple sugars, starch must be broken down into glucose before being
absorbed into the blood. In this way, the energy supplied by starchy foods such as pasta and
vegetables can be absorbed more slowly and be available for longer than other simple sugars.

How long before the onset of exercise should a meal be consumed? For most people, a meal high
in carbohydrates will be emptied from the stomach after three hours of fasting. As mentioned
earlier, exercising on an empty stomach is physiologically beneficial and eating three hours
before an exercise bout will usually give the stomach plenty of time to empty. Although the
circulating glucose provided by starchy foods is used as energy for working muscle to some
extent, most of the energy used comes from glycogen stores in the muscle itself. Therefore, it is
important to be eating high carbohydrate meals days before an event or exercise bout in order to
build up glycogen stores in the muscle and liver. Because of glycogen's branched structure and
hydrophilic hydroxyl groups, it is able to absorb significant quantities of water, which helps keep
the body hydrated during exercise.

Losing weight can also be related to carbohydrates. Fluctuations in hormones caused by the
ingestion of carbohydrates make the burning or utilization of fats more difficult. For example,
eating a meal high in carbohydrates will cause the release of insulin, a hormone that allows cells
to uptake glucose. However, insulin also acts on adipose tissue to inhibit the release of fatty acids
and promote their synthesis. Since most people are trying to become leaner by burning fat
through exercise, eating a meal before exercise may not be the best idea for losing weight. The
best time of the day to exercise with the intention of losing weight is in the morning. Since no
food has been consumed since the last meal of the previous day, levels of insulin in the blood
will be low. Furthermore, levels of glucagon will increase due to fasting and this hormone
stimulates the release of fatty acids from adipose tissue while inhibiting the breakdown of
glycogen from the liver. Epinephrine and Norepinephrine are also secreted in response to
exercise and these hormones mimic the effects of insulin by increasing availability of fatty acids
in the blood. Of course, fatty acids may only be utilized by working muscle under aerobic
conditions. Therefore, in order to burn fats effectively, the ratio of insulin to glucagons must be
low and the body must be performing exercise at a relatively low intensity for a prolonged period
of time.

It is true that some of the glucose ingested can be converted to fatty acids by the liver when there
is an excess of energy available to the body. But eating a diet low in carbohydrates is not a good
solution to a weight problem either. For example, many weight loss programs suggest diets that
are high in protein and low in carbohydrates. Besides high levels of ammonia in the body
through the breakdown of protein, a low carbohydrate diet will force the body into a condition
known as ketosis. Ketosis comes from the root word ketones which are produced in the liver
through the incomplete breakdown of fat. Although some of the amino acids that come from the
diet are gluconeogenic, meaning glucose producing, many amino acids are ketogenic and cannot
enter the citric acid cycle. Therefore, the level of ketones in the body rises while levels of
glucose fall and many organs begin to function less efficiently, including the brain, which relies
heavily on glucose. Furthermore, fats can only be metabolized when there is a certain amount of
glucose present to produce oxaloacetate that condenses with acetyl CoA in the citric acid cycle.
Since fatty acids are degraded directly to acetyl CoA, they cannot be used as an energy source.
Instead, they too are transformed in ketones. Although many tissues can utilize ketones for
energy, high levels in the blood stream are dangerous and low amounts of glucose in the blood
can be detrimental to the brain.

In addition, low carbohydrate diets without exercise will slow down metabolism. Weight loss
may be realized but the type of weight that is actually lost may include muscle mass as well as
fat. With less lean body mass, resting metabolic rates will decrease since skeletal muscle requires
more energy at rest and during exercise than adipose tissue. In conclusion, exercising during
fasting periods and eating a diet high in carbohydrates will not only increase well being, but will
also help to lose weight more effectively. Monitoring the amount of carbohydrate intake with the
amount of exercise will help to refine weight loss.

Glycolysis

Glycolysis is the sequence of reactions that converts glucose into pyruvate with the concomitant
production of a relatively small amount of ATP. Glucose is the starting material and two
molecules of pyruvate are the end products of the pathway.
 Figure %: The Glycolytic Pathway.

Now that we have a general understanding of the broad topics of metabolism and respiration, we
will turn our discussion to more specific metabolic pathways that lead to the derivation of ATP.
In this SparkNote we will look at glycolysis, the metabolism of glucose, a digestive product of
carbohydrates found in many food products that we ingest.

Taking place in the cell cytoplasm, glycolysis actually comprises a series of nine steps involving
a number of intermediate structures and specific enzymes that help catalyze each reaction. In this
section, we will go through each of these reactions, learning the roles of the associated
intermediates and enzymes. (Note: specific knowledge of the nine steps of glycolysis is not
necessary for the AP Biology test. In regard to that test, this summary presents all information
necessary about glycolysis and the first two sections of this SparkNote can be skipped. The third
section on fermentation, however, will be covered on the AP test).

Over the course of glycolysis' nine steps, the 6-carbon molecule glucose is broken down to two
3-carbon pyruvate molecules. The reaction does not occur spontaneously: 2 ATP molecules must
be broken down to drive the splitting of glucose into the 2 pyruvates. However, in the course of
the breakdown of glucose, the glycolysis reaction produces four ATP, resulting in a net gain of
two ATP for the entire process. Glycolysis also results in the production of 2 NADH molecules,
which eventually play an important role in the production of additional ATP in the electron
transport chain. Glycolysis itself is an anaerobic process. After a cell has completed glycolysis,
and depending on the circumstances in which the cell finds itself, that cell can either move into
the process of aerobic respiration and commence the citric acid cycle or continue with less
efficient aneorobic respiration in a process called fermentation.
There are two stages. The first involves the phosphorylation of the glucose ring in preparation for
an eventual breakdown into two 3-carbon molecules. In the second stage, the two 3-carbon
molecules are converted into pyruvate.

Definition of terms
Adenisine Triphosphate (ATP) - The molecule from which cells derive energy. Comprised of
an adenisine molecule bonded to three phosphates, each phosphate bond contains energy,
especially the third bond. By breaking that one bond and reducing ATP to adenisine diphosphate
(ADP), the cell can get the energy to carry out its various processes.
Alcohol dehydrogenase - The glycolytic enzyme responsible for catalyzing the reaction that
converts acetaldehyde to ethanol in the alcoholic fermentation of pyruvate.
Alcoholic fermentation - The process that converts pyruvate to carbon dioxide and ethanol that
takes place in yeast under anaerobic conditions.
Aldolase - The glycolytic enzyme responsible for catalyzing the reaction that converts fructose-
1,6-bisphosphate to glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phoshate
(DHAP).
Glucose - A six-carbon molecule that is a digestive product of carbohydrates and plays a major
role in the metabolism of living cells.
Glyceraldehyde-3-phosphate dehydrogenase - The glycolytic enzyme responsible for
converting glyceraldehyde-3-phosphate into 1,3-bisphoglycerate with the reduction of NAD to
NADH.
Hexokinase - The glycolytic enzyme responsible for catalyzing the reaction that transfers a
phosphate group from a molecule of ATP to a 6-membered glucose ring.
Homolactic fermentation - The process that converts pyruvate into lactate while oxidizing
NADH to NAD under anaerobic conditions.
Lactate dehydrogenase - The glycolytic enzyme responsible for converting pyruvate into
lactate while oxidizing NADH to NAD under anaerobic conditions.
Phosphofructokinase - The glycolytic enzyme responsible for converting fructose-6-phosphate
to fructose-1,6-bisphosphate by transferring a phosphate group from ATP to fructose-6-
phosphate.
Phosphoglucose isomerase - The glycolytic enzyme responsible for converting glucose-6-
phosphate to fructose-6-phosphate.
Phosphoglycerate kinase - The glycolytic enzyme responsible for converting 1,3-
bisphoglycerate into 3- phosphoglycerate by transferring a phosphate group from 1,3-
bisphoglycerate to an ADP molecule to yield ATP.
Phosphoglycerate mutase - The glycolytic enzyme responsible for rearranging 3-
phosphoglycerate into 2- phosphoglycerate.
Phosphorylation - A reaction that transfers a phosphate group from one molecule to another.
Pyruvate - The end product of glycolysis.
Pyruvate decarboxylase - The glycolytic enzyme responsible for converting pyruvate into
acetaldehyde under anaerobic conditions during alcoholic fermentation.
Triosphosphate isomerase - The glycolytic enzyme responsible for converting
dihydroxyacetone adenine phosphate into glyceraldehyde-3-phosphate so that it can continue in
the glycolytic pathway.

Stage 1: Glucose Breakdown

Glycolysis involves nine distinct reactions that convert glucose into pyruvate. In this section, we
will cover the first four of these reactions, which convert glucose into glyceraldehyde-3-
phosphate. Glucose is a six- memebered ring molecule found in the blood and is usually a result
of the breakdown of carbohydrates into sugars. It enters cells through specific transporter
proteins that move it from outside the cell into the cell's cytosol. All of the glycolytic enzymes
are found in the cytosol.

Step 1: Hexokinase

In the first step of glycolysis, the glucose ring is phosphorylated. Phosphorylation is the process
of adding a phosphate group to a molecule derived from ATP. As a result, at this point in
glycolysis, 1 molecule of ATP has been consumed.

Figure %: Step 1.

The reaction occurs with the help of the enzyme hexokinase, an enzyme that catalyzes the
phosphorylation of many six-membered glucose-like ring structures. A kinase is the name given
to an enzyme that phosphorylates other molecules. Atomic magnesium (Mg) is also involved to
help shield the negative charges from the phosphate groups on the ATP molecule. The result of
this phosphorylation is a molecule called glucose-6-phosphate (G6P), thusly called because the 6'
carbon of the glucose acquires the phosphate group.

Step 2: Phosphoglucose Isomerase

The second step of glycolysis involves the conversion of glucose-6-phosphate to fructose-6-

phosphate (F6P). This reaction occurs with the help of the enzyme phosphoglucose isomerase
(PI). As the name of the enzyme suggests, this reaction involves an isomerization reaction.

Figure %: Step 2.
The reaction involves the rearrangement of the carbon-oxygen bond to transform the six-
membered ring into a five-membered ring. To rearrangement takes place when the six-membered
ring opens and then closes in such a way that the first carbon becomes now external to the ring.

Step 3: Phosphofructokinase

In the third step of glycolysis, fructose-6-phosphate is converted to fructose- 1,6-bisphosphate

(FBP). Similar to the reaction that occurs in step 1 of glycolysis, a second molecule of ATP
provides the phosphate group that is added on to the F6P molecule.

Figure %: Step 3.

The enzyme that catalyzes this reaction is phosphofructokinase (PFK). As in step 1, a magnesium
atom is involved to help shield negative charges.

Step 4: Aldolase

The final step of the first stage of glycolysis utilizes the enzyme aldolase, which catalyzes the
cleavage of FBP to yield two 3-carbon molecules. One of these molecules is called
glyceraldehyde-3-phosphate (GAP) and the other is called dihydroxyacetone phosphate (DHAP).

Figure %: Step 4.
GAP is the only molecule that continues in the glycolytic pathway. As a result, all of the DHAP
molecules produced are further acted on by the enzyme triphoshpate isomerase (TIM), which
reorganizes the DHAP into GAP so it can continue in glycolysis. At this point in the glycolytic
pathway, we have two 3-carbon molecules, but have not yet fully converted glucose into
pyruvate

Stage 2: Conversion to Pyruvate

In this section, we will look at the reactions that convert our two 3-carbon molecules of
glyceraldehyde-3-phosphate (GAP) into pyruvate, the product of glycolysis. This conversion
occurs in five steps that we will review below. At this point, we will also see where oxygen
comes into play in glycolysis so that in the next section, we can look at the differences between
aerobic and anaerobic glycolysis. Keep in mind in this section that since we have split our 6-
carbon molecule into two 3-carbon molecules, each of these reactions is occurring in both of the
3-carbon molecules.

Step 5: Glyceraldehyde-3-phosphate Dehydrogenase

In this step, two main events take place: 1) glyceraldehyde-3-phosphate is oxidized by the
coenzyme nicotinamide adenine dinucleotide (NAD); 2) the molecule is phosphorylated by the
addition of a free phosphate group. The enzyme that catalyzes this reaction is glyceraldehyde-3-
phosphate dehydrogenase (GAPDH).

Figure %: Step 5.

The chemistry that takes place in this reaction is more complex than that of the previous
reactions we've discussed. Knowledge of organic chemistry is needed to understand the specific
mechanisms of the conversion. Generally, the enzyme GAPDH contains appropriate structures
and holds the molecule in a conformation such that it allows the NAD molecule to pull a
hydrogen off the GAP, converting the NAD to NADH. The phosphate group then attacks the
GAP molecule and releases it from the enzyme to yield 1,3 bisphoglycerate, NADH, and a
hydrogen atom. We will come back to the role of this NAD/NADH molecule in the next section.

Step 6: Phosphoglycerate Kinase

In this step, 1,3 bisphoglycerate is converted to 3-phosphoglycerate by the enzyme

phosphoglycerate kinase (PGK). This reaction involves the loss of a phosphate group from the
starting material. The phosphate is transferred to a molecule of ADP that yields our first molecule
of ATP. Since we actually have two molecules of 1,3 bisphoglycerate (because there were two 3-
carbon products from stage 1 of glycolysis), we actually synthesize two molecules of ATP at this
step. With this synthesis of ATP, we have cancelled the first two molecules of ATP that we used,
leaving us with a net of 0 ATP molecules up to this stage of glycolysis.
 Figure %: Step 6.

Again, we see that an atom of magnesium is involved to shield the negative charges on the
phosphate groups of the ATP molecule.

Step 7: Phosphoglycerate Mutase

This step involves a simple rearrangement of the position of the phosphate group on the 3
phosphoglycerate molecule, making it 2 phosphoglycerate. The molecule responsible for
catalyzing this reaction is called phosphoglycerate mutase (PGM). A mutase is an enzyme that
catalyzes the transfer of a functional group from one position on a molecule to another.

Figure %: Step 7.

The reaction mechanism proceeds by first adding an additional phosphate group to the 2' position
of the 3 phosphoglycerate. The enzyme then removes the phosphate from the 3' position leaving
just the 2' phosphate, and thus yielding 2 phsophoglycerate. In this way, the enzyme is also
restored to its original, phosphorylated state.

Step 8: Enolase

The eighth step involves the conversion of 2 phosphoglycerate to phosphoenolpyruvate (PEP).

The reaction is catalyzed by the enzyme enolase. Enolase works by removing a water group, or
dehydrating the 2 phosphoglycerate. The specificity of the enzyme pocket allows for the reaction
to occur through a series of steps too complicated to cover here.

Figure %: Step 8.

Step 9: Pyruvate Kinase

The final step of glycolysis converts phosphoenolpyruvate into pyruvate with the help of the
enzyme pyruvate kinase. As the enzyme's name suggests, this reaction involves the transfer of a
phosphate group. The phosphate group attached to the 2' carbon of the PEP is transferred to a
molecule of ADP, yielding ATP. Again, since there are two molecules of PEP, here we actually
generate 2 ATP molecules.

Figure %: Step 9.

We have now completed our discussion of the steps of glycolysis. If we go back and take count
of our ATP usage and generation, we find that we have consumed two molecules of ATP and
generate four to leave a net gain of two ATP molecules from the glycolytic pathway. We have
gone from our starting product, glucose, to our final product, pyruvate.

Anaerobic Respiration: Homolactic Fermentation

After Glycolysis

Glycolysis, as we have just described it, is an anaerobic process. None of its nine steps involve
the use of oxygen. However, immediately upon finishing glycolysis, the cell must continue
respiration in either an aerobic or anaerobic direction; this choice is made based on the
circumstances of the particular cell. A cell that can perform aerobic respiration and which finds
itself in the presence of oxygen will continue on to the aerobic citric acid cycle in the
mitochondria. If a cell able to perform aerobic respiration is in a situation where there is no
oxygen (such as muscles under extreme exertion), it will move into a type of anaerobic
respiration called homolactic fermentation. Some cells such as yeast are unable to carry out
aerobic respiration and will automatically move into a type of anaerobic respiration called
alcoholic fermentation.
 Figure %: Anaerobic vs. Aerobic pathways

More specifically, the differences in aerobic and anaerobic respiration rest on the different very
roles played by the NADH molecule produced in step 5 of glycolysis. In both aerobic and
anaerobic respiration, the NADH molecule is part of the enzyme complex and must be restored
to its NAD, oxidized state. If there are aerobic conditions, meaning oxygen is available, the
NADH molecule can be transported to the mitochondria where it can be immediately converted
back to NAD and plays a role in the electron transport chain. However, under anaerobic, oxygen-
deficient conditions, NADH gets converted back to NAD through anaerobic mechanisms,
whether homolactic or alcoholic fermentation.

Homolactic Fermentation

Instead of being immediately reoxidized after glycolysis step 5 as it would in aerobic respiration,
the NADH molecule remains in its reduced form until pyruvate has been formed at the end of
glycolysis. The pyruvate product of glycolysis gets further acted upon under anaerobic
conditions by the enzyme lactate dehydrogenase (LDH).

Figure %: Homolactic Fermentation.

In this reaction, the hydrogen from the NADH molecule is transferred to the pyruvate molecule.
This results in its carbon-oxygen double bond being reduced to a carbon-oxygen single bond
with the addition of a hydrogen atom. The result is the molecule lactate. From the lactate
product, lactic acid can be formed, which causes the muscle fatigue that accompanies strenuous
workouts where oxygen becomes deficient.
Alcoholic Fermentation

There is another way that the NADH molecule can be re-oxidized. Anaerobic conditions in yeast
convert pyruvate to carbon dioxide and ethanol. This occurs with the help of the enzyme
pyruvate decarboxylase which removes a carbon dioxide molecule from the pyruvate to yield an
acetaldehyde. The acetaldehyde is then reduced by the enzyme alcohol dehydrogenase which
transfers the hydrogen from NADH to the acetaldehyde to yield NAD and ethanol. This enzyme
is not found in humans.

Figure %: Alcoholic Fermentation.

Anaerobic Byproducts

As you can see, both of these anaerobic conditions leads to glycolytic products other than
pyruvate. These different products are necessary because the NADH molecule must be
reoxidized so that it can function in the next round of glycolysis of newly introduced glucose. If
oxygen is not present to help oxidize it, other reactions, such as those of homolactic and
alcoholic fermentation, must occur.

Glycolsis hetexose monophosphetic pathways are shown below