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Unit 8 – METABOLISM
Compiled by: Joselito R. Tumulak Jr., RChT, MS (cand.)
Biochemistry Professor

INTENDED LEARNING OUTCOMES

At the end of this unit, you will be able to:
1. Explain how cells manage the conflicting demands between anabolism and catabolism;
2. Relate the structure of common intermediate metabolites to its role in metabolism;
3. Describe the reactions, inhibition, and regulation of Krebs cycle;
4. Reason out how bulk ATP is produced in ETC;
5. Calculate the amount of ATP produced in the common catabolic pathways;
6. Describe the action of different stages of digestion on dietary carbohydrates, lipids and
proteins;
7. Describe the reactions, regulations and energy yield of glycolysis;
8. Describe the salient features of the other metabolic pathways that carbohydrates undergo,
specifically glycogenesis, glycogenolysis, gluconeogenesis, and pentose phosphate
pathway in terms of utilization of glucose, its role in biological systems, and energy
involved;
9. Calculate the ATP yield in β-oxidation of fatty acids and compare it with the energy yield
of carbohydrate catabolism;
10. Relate the production and significance of ketone bodies to β-oxidation; and
11. Describe reactions that utilize amino acids as energy source and how its nitrogen is
excreted.

UNIT OUTLINE
Topic Page
I. Overview
A. Nature of Metabolism 2
B. Key Intermediates of Metabolism
C. Foods That Are Basis of Human Nutrition
II. Biochemical Energy Production
A. Stages of Biochemical Energy Production
B. Digestion of the Three Basic Food Group 10
C. Citric Acid Cycle (Krebs Cycle)
D. Electron Transport Chain and Oxidative Phosphorylation
III. Metabolism of Carbohydrates, Lipids, and Proteins
A. Glycolysis
B. Other Metabolic Pathways of Carbohydrates
i. Gluconeogenesis
ii. Glycogen Metabolism
31
• Glycogenesis
• Glycogenolysis
iii. Pentose Phosphate Pathway
iv. Hormonal Controls of Carbohydrate Metabolism
C. Storage and Mobilization of Fats

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D. Catabolism of Glycerol
E. β-oxidation of Fatty Acids
F. Ketone Bodies
G. Amino Acid Utilization
i. Catabolism of the Nitrogen
ii. Fates of the Amino Acid Carbon Skeleton
CONCLUSION 60

I. OVERVIEW

A. Nature of Metabolism

Metabolism is the sum total of all the biochemical reactions that take place in a living organism.
Metabolic reactions fall into one of two subtypes: catabolism and anabolism.
• Catabolism is all metabolic reactions in which large biochemical molecules are broken
down to smaller ones. Catabolic reactions usually release energy. The reactions
involved in the oxidation of glucose are catabolic.
• Anabolism is all metabolic reactions in which small biochemical molecules are joined
together to form larger ones. Anabolic reactions usually require energy in order to
proceed. The synthesis of proteins from amino acids is an anabolic process.

Anabolism and catabolism are not mutually exclusive; they occur simultaneously in the cell.
The conflicting demands of concomitant catabolism and anabolism are managed by cells in 2
ways:
1. The cell maintains tight and separate regulation of both catabolism and anabolism,
so metabolic needs are served in an immediate and orderly fashion.
2. Competing metabolic pathways are often localized within different cellular
compartments. Isolating opposing activities in different compartments, such as
separate organelles, avoid interference between them. For example, the enzymes
for the catabolism of fatty acids are localized in the mitochondria, while
biosynthesis of fatty acids occur in the cytosol.

• There are other metabolic pathways in which their metabolites have dual purposes – they
serve both in catabolism and anabolism. These pathways are considered amphibolic.

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Metabolic Pathways
The metabolic reactions that occur in a cell are usually organized into sequences called
metabolic pathways. A metabolic pathway is a series of consecutive biochemical reactions used
to convert a starting material into an end product. Such pathways may be linear, in which a series
of reactions generates a final product, or cyclic, in which a series of reactions regenerates the
first reactant.

The major metabolic pathways for all life forms are similar. This enables scientists to study
metabolic reactions in simpler life forms and use the results to help understand the corresponding
metabolic reactions in more complex organisms, including humans.

Metabolism and Cell Structure

Knowledge of the major structural features of a cell is a prerequisite to understanding where
metabolic reactions take place. Recall, that there are 2 types of cells – prokaryotic and eukaryotic.
We will focus our attention to the eukaryotic cells, as these are the types of cells present in
humans. A eukaryotic cell is a cell in which the DNA is found in a membrane-enclosed nucleus.
Cells of this type, which are found in all higher organisms, are about 1000 times larger than
bacterial cells. The key components in a eukaryotic cell are shown in the figure below - the outer
membrane, nucleus, cytosol, ribosomes, lysosomes, and mitochondria.

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• The cytoplasm is the water-based material of a eukaryotic cell that lies between the
nucleus and the outer membrane of the cell. Within the cytoplasm are several kinds of
small structures called organelles. An organelle is a minute structure within the
cytoplasm of a cell that carries out a specific cellular function. The organelles are
surrounded by the cytosol. The cytosol is the water-based fluid part of the cytoplasm of
a cell.
• Ribosomes are the sites where protein synthesis occurs.
• A lysosome is an organelle that contains hydrolytic enzymes needed for cellular
rebuilding, repair, and degradation. Some lysosome enzymes hydrolyze proteins to amino
acids; others hydrolyze polysaccharides to monosaccharides. Bacteria and viruses
“trapped” by the body’s immune system are degraded and destroyed by enzymes from
lysosomes.
• A mitochondrion is an organelle that is responsible for the generation of most of the
energy for a cell. Much of the discussion of catabolism deals with the energy-producing
chemical reactions that occur within mitochondria.
o Anatomy of Mitochondrion:
§ Mitochondria are sausage-shaped organelles containing both an outer
membrane and a multifolded inner membrane. The outer membrane,
which is about 50% lipid and 50% protein, is freely permeable to small
molecules. The inner membrane, which is about 20% lipid and 80%
protein, is highly impermeable to most substances.
§ The non-permeable nature of the inner membrane divides a mitochondrion
into two separate compartments—an interior region called the matrix and
the region between the inner and outer membranes, called
the intermembrane space.
§ The folds of the inner membrane that protrude into the matrix are
called cristae.

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B. Key Intermediates of Metabolism

1. Adenosine Phosphates (ATP, ADP, and AMP)

Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine
monophosphate (AMP) are nucleotides. All three of these molecules contain the adenine and
β-D-ribose joined together by a β-N-glycosidic bond, forming adenosine. These molecules
differ only on the number of phosphate groups they contain. Block diagrams and structural
formulas for these three adenosine phosphates follow.

In all three molecules, the first phosphate group is attached to the ribose by a phosphoric ester
bond. In the case of ADP and ATP, the next phosphate groups are attached by phosphoric
anhydride bonds. The figure above shows actual structural formulas for these three adenosine
phosphates. Highlighted within the structural formulas are the bonds that the phosphate groups
participate in.

A phosphoric anhydride bond contains more chemical energy than a phosphoric ester
linkage. Thus, when ATP and ADP are hydrolyzed to form phosphate ions, they release more
energy per phosphate group than does AMP. Conversely, when inorganic phosphate bonds to
AMP or ADP, greater amounts of energy are added to the chemical bond than when it bonds to
adenosine. ATP releases the most energy. This property makes ATP a very useful compound
for energy storage and release. The energy gained from food is stored in the form of ATP.

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2. Flavin Adenine Dinucleotide (FAD)

Flavin adenine dinucleotide (FAD) is one of the coenzymes required in metabolic redox
reactions. The block diagram and actual structural formula of FAD is shown below:

The flavin and ribitol subunits in this structure together constitute vitamin B2 (riboflavin). The
coenzyme FAD is thus one of the biochemically active forms of riboflavin; the activating factor
is the ADP subunit. In metabolism, the operative part of FAD is the flavin portion. It undergoes
reduction by gaining 2 H+ and 2 e- as shown below. For this reason, the reduced form of FAD is
often referred to as FADH2.

A typical cellular reaction in which FAD serves as the oxidizing agent involves a -CH2-CH2-
portion of a substrate being oxidized to produce a carbon–carbon double bond.

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3. Nicotinamide Adenine Dinucleotide (NAD+)

Nicotinamide adenine dinucleotide (NAD+) also has coenzyme functions in metabolic redox
pathways, like FAD. The structure of NAD+ is illustrated below.

The positive charge in the notation for the oxidized form of NAD+ is due to the positive charge in
the pyrimidine nitrogen of its nicotinamide portion. Just like FAD, it also has a B vitamin moiety,
specifically nicotinamide, an amide derivative of vitamin B3 (niacin). In metabolism, the
operative part of NAD+ is the nicotinamide portion. It undergoes reduction gaining 2 e-, but
unlike the flavin of FAD, only 1 of the H+ in the reduction reaction is gained. For that reason, the
reduced form of NAD+ is referred to as NADH.

A typical cellular reaction in which NAD+ serves as the oxidizing agent is the oxidation of a
secondary alcohol to give a ketone.

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Because both NAD+ and FAD coenzymes can be reduced gaining H+ and e-, they are transporter
molecules of H+ and e-.

4. Coenzyme A (coA)
The principal compound in the common catabolic pathway is coenzyme A (coA). This is an
acetyl (CH3CO-)-transporting molecule. This is a derivative of the vitamin B5 (pantothenic
acid). The block diagram and actual structural formula of coenzyme A is shown below:

The operative portion of coenzyme A is the sulfhydryl group in the ethanethiol subunit of the
coenzyme. As mentioned, this coenzyme functions as acetyl group-transporter in metabolic
pathways. This is where the acetyl group will attach to.

Recall that an acetyl group is the portion of an acetic acid molecule that remains after the -OH
group is removed from the carboxyl carbon atom. An acetyl group bonds to CoA through a
thioester bond to give acetyl CoA.

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C. Food Substances That Are Basis of Human Nutrition

The use of food by organisms is termed nutrition. The ability of an organism to use a particular
food material depends upon its chemical composition and upon the metabolic pathways available
to the organism. Food includes macronutrients – proteins, carbohydrates, and lipids – and the
micronutrients – vitamins and minerals. In addition, some essential fibers are also required in
the diet.

Proteins
Humans must consume proteins to make new proteins, especially those protein food
containing essential amino acids that we are not capable of synthesizing in adequate amount
in the body. Excess dietary protein is then merely a source of metabolic energy. Some of these
amino acids can be converted to glucose (termed glucogenic), whereas others can be converted
to fatty acids and/or ketone bodies (termed ketogenic). If fat and carbohydrate are already
adequate for the energy needs of an individual, then both kinds of amino acids will be converted
to triacylglycerol and stored in adipose tissues.

An individual’s protein undergoes a constant process of degradation and resynthesis. Together

with dietary protein, this recycled protein material participates in a nitrogen equilibrium, or
nitrogen balance.
• A positive nitrogen balance occurs whenever there is a net increase in the organism’s
protein content, such as during periods of growth.
• A negative nitrogen balance exists when dietary intake of nitrogen is insufficient to meet
the demands for new protein synthesis.

Carbohydrates
The principal purpose of carbohydrate in the diet is production of metabolic energy. Simple
sugars are metabolized in the glycolytic pathway. Complex carbohydrates are degraded into
simple sugars, which then can enter the glycolytic pathway. Carbohydrates are also essential
components of nucleotides, nucleic acids, glycoproteins, and glycolipids.

Human metabolism can adapt to a wide range of dietary carbohydrate levels, but the brain
requires glucose for fuel. When dietary carbohydrate consumption exceeds the energy needs of
the individual, excess carbohydrate is converted to triacylglycerols and glycogen for long-term
energy storage. On the other hand, when dietary carbohydrate intake is low, ketone bodies are
formed from acetate units to provide metabolic fuel for the brain and other organs.

Lipids
Fatty acids and triacylglycerols can be used as fuel by many tissues in the human body, and
phospholipids are essential components of all biological membranes. Even though the human
body can tolerate a wide range of fat intake levels, there are disadvantages in either extreme.
Excess dietary fat is stored as triacylglycerols in adipose tissue, but high levels of dietary fat
can also increase the risk of atherosclerosis and heart disease. Moreover, high dietary fat levels
are also correlated with increased risk for colon, breast, and prostate cancers.

When dietary fat consumption is low, there is a risk of essential fatty acid deficiencies. The
human body cannot synthesize linoleic acid and linolenic acid, so these must be acquired in

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the diet. In addition, arachidonic acid can by synthesized in humans only from linoleic acid, so it
too is classified as essential. The essential fatty acids are key components of biological
membranes, and arachidonic acid is the precursor to prostaglandins, which mediate a variety of
processes in the body.

Fibers
The components of food materials that cannot be broken down by human digestive enzymes are
referred to as dietary fiber. There are several kinds of dietary fiber, each with its own chemical
and biological properties.
• Cellulose and hemicellulose are insoluble fiber materials that stimulate regular function
of the colon. They may play a role in reducing the risk of colon cancer.
• Lignins, another class of insoluble fibers, absorb organic molecules in the digestive
system. Lignins bind cholesterol and clear it from the digestive system, reducing the risk
of heart disease.
• Pectins and gums are water-soluble fiber materials that form viscous gel-like
suspensions in the digestive system, slowing the rate of absorption of many nutrients,
including carbohydrates, and lowering serum cholesterol in many cases.

The insoluble fibers are prevalent in vegetable grains. Water-soluble fiber is a component of fruits,
legumes, and oats.

II. BIOCHEMICAL ENERGY PRODUCTION

A. Stages of Biochemical Energy Production

The energy needed to run the human body is obtained from ingested food through a multistep
process that involves several different catabolic pathways. There are four general stages in the
biochemical energy production process, and numerous reactions are associated with each stage.

Stage 1:
The first stage, digestion, begins in the mouth (saliva contains starch-digesting enzymes),
continues in the stomach (gastric juices), and is completed in the small intestine (location of most
digestive enzymes and bile salts). The end products of digestion—glucose and other
monosaccharides from carbohydrates, amino acids from proteins, and fatty acids and glycerol
from fats and oils—are small enough to pass across intestinal membranes and into the blood,
with the aid of membrane transport systems. Once in the blood, they are then distributed to the
cells in various parts of the body.

Stage 2:
The second stage, acetyl group formation, involves numerous reactions, some of which occur
in the cytosol of cells and some in cellular mitochondria. The small molecules from digestion are
further oxidized during this stage. Primary products include two-carbon acetyl units (which
become attached to coenzyme A to give acetyl CoA) and reduced coenzymes.

Stage 3:
The third stage, the citric acid cycle, occurs inside mitochondria. Here acetyl groups are oxidized
to produce and energy. Some of the energy released by these reactions is lost as heat, and some

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is carried by the reduced coenzymes NADH and FADH2 to the fourth stage. The CO2 that is
exhaled as part of the breathing process comes primarily from this stage.

Stage 4:
The fourth stage, the electron transport chain and oxidative phosphorylation, also occurs
inside mitochondria. NADH and FADH2 supply the “fuel” (hydrogen ions and electrons) needed to
produce ATP molecules, the primary energy carriers in metabolic pathways. Molecular O2,
inhaled via breathing, is converted to H2O in this stage.

The reactions in stages 3 and 4 are the same for all types of foods (carbohydrates, fats, proteins).
These reactions constitute the common metabolic pathway, which is the sum total of the
biochemical reactions of the citric acid cycle, the electron transport chain, and oxidative
phosphorylation. The reactions of the common metabolic pathways are linked together.

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B. Digestion of the Three Basic Food Groups

Digestion and Absorption of Carbohydrates

The figure below summarizes the digestion and absorption of carbohydrates.

1. The digestion of carbohydrates begins in the mouth (pH 6.2-7.6), where the
enzyme salivary α-amylase catalyzes the hydrolysis of α-glycosidic linkages in starch
from plants and glycogen from meats to produce smaller polysaccharide dextrin and
some disaccharide maltose. At this point, disaccharides do not undergo chemical
digestion as no enzymes capable of hydrolyzing them are secreted by the organs in the
oral cavity.
2. Only a small amount of carbohydrate digestion occurs in the mouth because food is
swallowed so quickly, and the salivary α -amylase is inactivated by the acidic
environment of the stomach (pH 1.5-3.5), and the stomach’s own secretions do not
contain any carbohydrate-digesting enzymes.
3. The primary site for carbohydrate digestion is within the small intestine, where α-
amylase, this time secreted by the pancreas, again begins to function. Prior to further
digestion, the acidic content of the stomach is neutralized by bicarbonate ions which are
also secreted by the pancreas. Pancreatic α-amylase breaks down polysaccharide
chains into shorter and shorter segments until the disaccharide maltose is the dominant
species. At this stage, the carbohydrate mixture present is a disaccharide mixture of
lactose (originally present as such), sucrose (originally present as such), and maltose
(obtained from polysaccharide breakdown).
4. The final step in carbohydrate digestion occurs on the outer membranes of intestinal
mucosal cells, where the disaccharidases, enzymes that convert disaccharides to
monosaccharides, are located. The important disaccharidases are maltase,
sucrase, and lactase. These enzymes convert, respectively, maltose to two glucose
units, sucrose to one glucose and one fructose unit, and lactose to one glucose and
one galactose unit.
5. The three major breakdown products from carbohydrate digestion are thus glucose,
galactose, and fructose. These monosaccharides are absorbed into the bloodstream
through the intestinal wall which are lined with fingerlike projections called villi, which

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are rich in blood capillaries. Absorption is by active transport, which needs ATP. Protein
carriers mediate the passage of the monosaccharides through cell membranes.
6. After their absorption into the bloodstream, monosaccharides are transported to the liver,
where fructose and galactose are rapidly converted into compounds that are
metabolized by the same pathway as glucose.

Digestion and Absorption of Fats

The figure below summarizes the digestion and absorption of fats:

1. Triacylglycerols are insoluble in water, just like any other lipids. Hence water-based
salivary enzymes in the mouth have little effect on them. In the mouth, no fat digestion
will happen due to the absence of enzymes that can hydrolyze triacylglycerol.
2. Chewed food travels to the stomach. The churning action of the stomach breaks up
triacylglycerol materials into small globules, or droplets, which float as a layer above the
other components of swallowed food. The resulting material is called chyme, a thick semi-
liquid material made up of small triacylglycerol globules, other partially digested food,
and gastric secretions (hydrochloric acid and several enzymes). When we eat high fat
food, it stays longer in the stomach. High fat food needs more churning. Chemical
digestion of fats also begins in the stomach. Under the action of gastric lipase enzymes,
hydrolysis of triacylglycerols occurs. Normally, about 10% of TAGs undergo hydrolysis in
the stomach, but regular consumption of a high-fat diet can induce the production of higher
levels of gastric lipases.
3. From the stomach, the chyme travels to the small intestine and cholecystokinin signals
the release of bile stored in the gall bladder. The bile emulsifies the chime. Colloid
particle formation through bile emulsification “solubilizes” the triacylglycerol globules, and
digestion of the TAGs resumes. The major enzymes involved at this point are the
pancreatic lipases, which hydrolyze ester linkages between the glycerol and fatty acid
units of the TAGs. Complete hydrolysis does not usually occur; only two of the three
fatty acid units are liberated, producing a monoacylglycerol and two free fatty acids.
Occasionally, enzymes remove all three fatty acid units, leaving a free glycerol molecule.

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4. The hydrolysis products, with the help of bile, will form micelles. Micelles are way smaller
than the original fat droplets. Because of this, micelles can be absorbed easily by the
intestinal cell membrane, where they will be repackaged as triacylglycerols again.
These triacylglycerols will combine with membrane lipids and water-soluble proteins to
produce lipoproteins, which are called chylomicrons. Two hours after meal, the
concentration of chylomicron begins to rise, and it reaches its peak at 4 to 6 hours after
meal. It can go to the liver for processing or be stored in the adipose tissues.
5. Chylomicrons will enter the lymphatic system to exit into the blood stream via the
thoracic duct.
6. In the blood stream, chylomicrons release TAGs using the lipoprotein lipase, which are
in the lining of the blood vessels in the muscles that uses fatty acids as fuel and fat
synthesis. Fatty acids and glycerol then absorbed by the cells can either be broken down
into acetyl CoA or stored as lipids.

Digestion and Absorption of Proteins

The figure below illustrates the stages in protein digestion and absorption:

1. Although proteins are well suited to the aqueous environment of the mouth, there are no
salivary enzymes that could hydrolyze chemical bonds in proteins.
2. Digestion of protein starts in the stomach where the gastrin, the hormone from stomach
mucosal cells, causes secretion of hydrochloric acid (HCl) and pepsinogen. HCl
maintains the pH of the stomach at pH 1.5-2.0, which helps kills bacteria, denature
globular proteins, and activates pepsinogen to pepsin. Pepsin can hydrolyze some of
the peptide bonds in proteins. At this point, proteins will be at least 10% hydrolyze to
large polypeptide chains, which will be transported to the small intestine in small batches.
3. In the small intestine, the passage of acidic proteins stimulates the production of the
hormone secretin, which stimulates the production of bicarbonate ion (HCO3-) to
neutralize the acidic proteins. Now, the pH will be about 7-8, which is ideal for the
pancreatic enzymes: trypsin, chymotrypsin and carboxypeptidase. These pancreatic
enzymes will work on the neutralized proteins and hydrolyze them. Intestinal mucosal
cells will secrete aminopeptidase to further hydrolyze the proteins and forming the amino
acid pool.

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4. Lastly, amino acid will be absorbed by the intestinal lining through active transport, and
then transported to the blood stream for distribution.

C. The Citric Acid Cycle

• Definition: The Citric Acid Cycle is the series of biochemical reactions in which the
acetyl portion of acetyl CoA is oxidized to carbon dioxide.
• Other by-products: Reduced coenzymes FADH2 and NADH, and GTP
• Other names: Krebs Cycle (based from its discoverer, Hans Adolf Krebs) and
tricarboxylic acid (TCA) cycle (based on the fact that citric acid has 3 carboxyl groups)
• Location: Mitochondrial matrix where the needed enzymes are found, except the
succinate dehydrogenase which is integrated in the inner mitochondrial membrane.

Reactions of the Citric Acid Cycle

The Citric Acid cycle is a cyclic type of metabolic pathway and is composed of 8 reactions. The 2
most important reactions in the Krebs cycle is oxidation and decarboxylation. Oxidation, which
produces NADH or FADH2, is encountered in four of the eight steps, and decarboxylation,
wherein a carbon chain is shortened by the removal of a carbon atom as a molecule, is
encountered in two of the eight steps.

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The Citric Acid Cycle can be divided into 2 phases. The first 5 reactions are for (1) production of
2 CO2 molecules for acetyl coA and the next 3 reactions are for (2) regeneration of
oxaloacetate.

Step 1: Formation of Citrate

The first reaction is the formation of citrate with the help of the enzyme citrate synthase. Here 2-
carbon acetyl of acetyl coA reacts with 4-carbon oxaloacetate to form 6-carbon citrate.

• Regulation: The enzyme of the reaction, citrate synthase, is highly regulated by

NADH and succinyl coA which are both products of the citric acid cycle. They serve
as allosteric inhibitor of this enzyme.

Step 2: Isomerization of Citrate to Isocitrate

The next step is to isomerize citrate to isocitrate with the aid of the enzyme aconitase.
• Reason: Citrate is a poor candidate for further oxidation because it contains
tertiary alcohol which is difficult to oxidized. Isocitrate, however has secondary
alcohol which is oxidizable.

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• Inhibitor: Fluoroacetate is an extremely poisonous substance that blocks the citric acid
cycle in vivo. For this reason, this is used as rodent poison. The action of fluoroacetate
has been traced to aconitase. Fluoroacetate readily crosses both the cellular and the
mitochondrial membranes, and in the mitochondria it is converted to fluoroacetyl coA by
acetyl coA synthetase. Fluoroacetyl coA can then condensed with oxaloacetate in the
presence of the enzyme citrate synthase to form fluorocitrate. This product then inhibits
the enzyme aconitase stopping the citric acid cycle.

Fluoroacetate may be viewed as Trojan horse inhibitor. Analogous to the giant Trojan
horse of Greek myth, which soldiers of Troy took into their city, not knowing the Greeks
soldiers were hidden inside and waiting to attack, fluoroacetate enters the citric acid cycle
innocently enough in the citrate synthase reaction until it is converted to fluorocitrate, a
potent inhibitor of aconitase.

Step 3: Oxidative Decarboxylation of Isocitrate to α-Ketoglutarate

In this step, isocitrate undergoes oxidative decarboxylation to form α-ketoglutarate, with
concomitant reduction of NAD+ to NADH using the enzyme isocitrate dehydrogenase.

NAD+ NADH + H+

This reaction is the first of the 2 reactions that produced CO2, and this also provides the first
connection of the citric acid cycle and the electron-transport chain and oxidative phosphorylation,
via its production of NADH. This reaction is also connected to protein metabolism, as α-
ketoglutarate is a crucial keto-acid for aminotransferase reactions.

• Regulation: Like the reaction of the first step, this reaction is also highly regulated.
NADH and ATP are allosteric inhibitors, whereas ADP acts as allosteric activator. The
enzyme is almost inactive in the absence of ADP.

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Step 4: Oxidative Decarboxylation of α-Ketoglutarate to Succinyl coA

In this step, a second oxidative decarboxylation occurs in α-ketoglutarate forming succinyl
coA with concomitant reduction of NAD+ to NADH. This involves the three-enzyme system, α-
ketoglutarate dehydrogenase complex. The B vitamin thiamine (B1), in the form of thiamine
pyrophosphate (TPP), is part of the enzyme complex, as is Mg2+ ion.

Step 5: Thioester Bond Cleavage in Succinyl CoA and Phosphorylation of GDP

For purposes of understanding the structural changes that occur, the reaction can be considered
to occur in two steps:
(1) succinyl CoA is converted to succinyl phosphate (a high-energy phosphate
compound) with coA as a by-product, followed by
(2) transfer of phosphoryl group in succinyl phosphate to GDP forming guanosine
triphosphate (GTP) and succinate
The entire reaction is catalyzed by the enzyme succinyl-CoA synthetase (also known as
succinate thiokinase).

The transfer of phosphoryl group to GDP is known as substrate-level phosphorylation, in

which a substrate provides the energy for phosphorylation, rather than an electron-transport
chain or proton gradient. The GDP that is produced can exchange its terminal phosphoryl group
with ADP in the presence of the enzyme nucleoside diphosphate kinase.

Take a pause!

This is a good point to pause in our trip down the citric acid cycle and see what has happened.
The acetyl moiety of acetyl coA was linked to oxaloacetate, and was later oxidized to liberate
2 molecules of CO2. The cycle has produced 2 molecules of NADH and 1 ATP thus far, and
has left a 4-carbon compound succinate. The citric acid cycle can now be completed by
converting succinate back to oxaloacetate.

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Step 6: Oxidation of Succinate to Fumarate

This step is the third oxidation reaction of the cycle. The enzyme involved is succinate
dehydrogenase, and the oxidizing agent is FAD rather than NAD+. Two hydrogen atoms are
removed from the succinate to produce fumarate, a species with a trans double bond. FAD is
reduced to FADH2 in the process.

In contrast with all of the other enzymes of the citric acid cycle, which are soluble proteins found
in mitochondrial matrix, succinate dehydrogenase is an integral membrane protein tightly
associated with the inner mitochondrial membrane.

Step 7: Hydration of Fumarate to L-Malate

In this step, the enzyme fumarase catalyzes the addition of water to the double bond of
fumarate. The enzyme is stereospecific, so only the L isomer of the product malate is produced.

Step 8: Oxidation of Malate Back to Oxaloacetate

In the last step of the citric acid cycle, L-malate is oxidized to oxaloacetate by malate
dehydrogenase with concomitant reduction of NAD+ to NADH. This reaction is reversible, and
the conversion of oxaloacetate back to L-malate is more favorable. The reaction is, however,
pulled forward by the favorable citrate synthase reaction.

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Summary of the Reactions of the Citric Acid Cycle

The reactions, important by-products, and the regulation and inhibition of each step is
summarized by the table below:
Step Substrate Product Enzyme By-Products Regulators/Inhibitors
1 oxaloacetate citrate citrate synthase Coenzyme A Allosteric Inhibitors:
• NADH
• succinyl coA
2 citrate isocitrate aconitase Inhibitor (external):
• fluoroacetate
3 isocitrate α- isocitrate CO2 Allosteric Inhibitor:
ketoglutarate dehydrogenase NADH (used in • NADH
ETC) • ATP
Allosteric Activator:
• ADP
4 α- succinyl coA α-ketoglutarate CO2
ketoglutarate dehydrogenase NADH (used in
complex ETC)
(requires
thiamine
pyrophosphate
(TPP) and Mg2+)
5 succinyl coA succinate succinyl coA GTP
synthetase (equivalent to
ATP)
6 succinate fumarate succinate FADH2 (used
dehydrogenase in ETC)
(an integral
membrane
enzyme, the only
enzyme not in
the matrix)
7 fumarate L-malate fumarase
8 L-malate oxaloacetate malate NADH (used in
dehydrogenase ETC)

The overall reaction of the citric acid cycle can be summarized by the general equation below:

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Important features of the cycle include the following:

1. The “fuel” for the cycle is acetyl CoA, obtained from the breakdown of carbohydrates,
fats, and proteins.
2. Four of the cycle reactions involve oxidation and reduction. The oxidizing agent is
either NAD+ (Steps 3, 4 & 8) or FAD (Step 6). The operation of the cycle depends on
the availability of these oxidizing agents. NAD+ is the oxidizing agent when a carbon–
oxygen double bond is formed; FAD is the oxidizing agent when a carbon–carbon double
bond is formed.
3. The 3 NADH and 1 FADH2 that are formed during the cycle carry electrons (e-) and H+ to
the electron transport chain through which ATP is synthesized.

4. Two carbon atoms enter the cycle as the acetyl moiety of acetyl CoA, and two carbon
atoms leave the cycle as two molecules of CO2. The carbon atoms that enter and
leave are not the same ones. The carbon atoms that leave during one turn of the cycle
are carbon atoms that entered during the previous turn of the cycle.
5. Four B vitamins are necessary for the proper functioning of the cycle: riboflavin (in both
FAD and the α-ketoglutarate dehydrogenase complex), niacin (in NAD+), pantothenic
acid (in coA), and thiamine (in the α-ketoglutarate dehydrogenase complex).
6. One high-energy GTP molecule is produced by substrate-level phosphorylation which is
equivalent to 1 ATP.
Regulation of the Citric Acid Cycle
The rate at which the citric acid cycle operates is controlled by the body’s need for energy (ATP).
When the body’s ATP supply is high, the ATP present inhibits the activity of citrate synthase,
the enzyme in Step 1 of the cycle. When energy is being used at a high rate, a state of low ATP
and high ADP concentrations, the ADP activates citrate synthase and the cycle speeds up. A
similar control mechanism exists at Step 3, which involves isocitrate dehydrogenase; here NADH
acts as an inhibitor and ADP as an activator.

Citric Acid Cycle is an Amphibolic Pathway

We have viewed the citric acid cycle thus far as a catabolic process because it oxidizes acetyl
to CO2 and converts the liberated energy to ATP and reduced coenzymes. However, citric acid
can be considered an amphibolic pathway as it is also involved in anabolism.

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The figure below shows that the four-, five- and six-carbon species produced in the citric acid
cycle also fuel a variety of biosynthetic processes. α-Ketoglutarate, succinyl coA, fumarate,
and oxaloacetate are all precursors of important cellular species.

1. A transamination reaction converts α-ketoglutarate directly to glutamate, which can

then serve as a versatile precursor for proline, arginine, and glutamine.
2. Succinyl coA provides most of the carbon atoms of the porphyrins.
3. Oxaloacetate can be transaminated to produce aspartate. Aspartate itself is a precursor
of the pyrimidine nucleotides and, in addition, is a key precursor for the synthesis of
asparagine, methionine, lysine, threonine, and isoleucine. Oxaloacetate can also be
decarboxylated to yield phosphoenolpyruvate (PEP), which is a key element of several
pathways, namely:
a. synthesis (in plants and microorganisms) of the aromatic amino acids
phenylalanine, tyrosine, and tryptophan;
b. formation of 3-phosphoglycerate and conversion to the amino acids serine,
glycine, and cysteine; and
c. gluconeogenesis, the pathway that synthesizes new glucose and many other
carbohydrates.

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D. Electron Transport Chain and Oxidative Phosphorylation

The electrons stored in the form of the reduced coenzymes, NADH or FADH2, are passed
through an elaborate and highly organized chain of proteins and coenzymes known as the
electron transport chain (ETC) finally reaching O2 (molecular oxygen), the terminal electron
acceptor. Each component of the chain can exist in 2 oxidation states, and each component is
successively reduced and re-oxidized as electrons move through the chain from NADH or FADH2
to O2. NADH and FADH2 are oxidized in this process.

Water is formed when the electrons and hydrogen ions that originate from these reactions react
with molecular oxygen.

In the course of electron transport, a proton gradient is established across the inner
mitochondrial membrane. It is the energy of this proton gradient that drives ATP synthesis in the
process known as oxidative phosphorylation.

Electron Transport Chain

The processes of electron transport are membrane associated. They are localized in the
mitochondria, which are also the sites of citric acid cycle activity and fatty acid oxidation. The
enzymes and electron carriers needed for the ETC are located along the inner mitochondrial
membrane. Within this membrane are four distinct protein complexes, each containing some of
the molecules needed for the ETC process to occur. These four protein complexes, which are
tightly bound to the membrane, are:
Complex I: NADH–coenzyme Q reductase
Complex II: Succinate–coenzyme Q reductase
Complex III: Coenzyme Q–cytochrome c reductase
Complex IV: Cytochrome c oxidase

The organization of the electron transport chain is illustrated on the next page. The electron
carriers, coenzyme Q (ubiquinone) and cytochrome c, which are not tightly associated with

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any of the four complexes, serve as mobile electron carriers that shuttle electrons between the
various complexes.

Complex I: NADH-coenzyme Q reductase

The sequence of electron-carrying enzyme systems starts with complex I. Complex I is the
largest of the complex in the ETC and it comprises 40 subunits, including the B-vitamin-containing
flavin mononucleotide (FMN) and several iron–sulfur proteins (FeSP).

The net result of electron movement through complex I is the transfer of electrons from NADH
to coenzyme Q (CoQ) resulting in the oxidation of NADH to NAD+ and oxidation of CoQ to
CoQH2.
NADH + H+ + CoQ à NAD+ + CoQH2
The actual electron transfer process is not, however, a single-step direct transfer of electrons from
NADH to CoQ. There several intermediate carriers are involved.
• The first electron transfer occurs when the electrons of NADH are transported into a flavin
mononucleotide (FMN) which is reduced to FMNH2. The NADH is converted to NAD+ in
the process.
• The next step involve transfer of electrons from the reduced FMNH2 through a series
of iron/sulfur proteins (FeSPs). The iron present in these FeSPs is ferric ion (Fe3+),
which is reduced to ferrous ion (Fe2+).
• In the final complex I reaction, Fe(II)SP units passes an electron to CoQ, changing it
from its oxidized form (CoQ) to its reduced form (CoQH2).
• The reduced CoQ transports the electrons to complex III. CoQ is lipid soluble and can
move laterally within the mitochondrial membrane.

The diagram below illustrates the transfer of electrons in the Complex (I):

Some of the energy released in Complex I reaction is used to move 4 H+ across the inner
membrane from the matrix to the intermembrane space.

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Complex II: Succinate–coenzyme Q reductase

Complex II is much smaller than complex I which contains only four subunits, including two
FeSPs. This complex is used to transfer the electrons to CoQ coming from the FADH2 that is
generated in the citric acid cycle when succinate is converted to fumarate.
FADH2+ CoQ à FAD+ CoQH2

CoQ is associated with the operations in complex II in a manner similar to its actions in complex
I. It is the final recipient of the electrons from FADH2, with iron–sulfur proteins serving as
intermediaries. The figure below shows the diagram for the electron transfer of in complex II.

Complexes I and II produce a common product, the reduced form of coenzyme Q (CoQH2). As
was the case with complex I, the reduced CoQH2 shuttles electrons to complex III. However,
unlike Complex I, Complex II does not transport H+ across the inner membrane from the matrix
to the intermembrane space.

Take a pause!

Note the general pattern that is developing for electron carriers present in the electron transport
chain. They are reduced (accept electrons) in one step and then are oxidized (lose electrons)
in the next step so that they are regenerated can again participate in electron transport chain
reactions.

Complex III: Coenzyme Q–cytochrome c reductase

Complex III receives the electrons carried by CoQ from Complex I and Complex II. It contains 11
different subunits. Electron carriers present include several FeSPs as well as several
cytochromes. A cytochrome is a heme-containing protein in which reversible oxidation and
reduction of an iron atom occur. Heme, a compound also present in hemoglobin and myoglobin,
has the structure:

Heme-containing proteins function similarly to FeSPs; iron changes back and forth between the
3+ and 2+ oxidation states.

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Various cytochromes, abbreviated cyt a, cyt b, cyt c, and so on, differ from each other in
(1) their protein constituents,
(2) the manner in which the heme is bound to the protein, and
(3) attachments to the heme ring.

The initial substrate for complex III is CoQH2 molecules carrying the electrons that have been
processed through complex I (from NADH) and also those processed through complex II (from
FADH2). The electron transfer process proceeds from CoQH2 to an FeSP, then to cyt b, then to
another FeSP, then to cyt c1, and finally to cyt c. The figure below shows diagrammatically the
electron transfer steps associated with complex III.

Cyt c is water-soluble and can move laterally in the intermembrane space; it delivers its
electrons to complex IV. Cyt c is the only one of the cytochromes that is water soluble.

Like Complex I, some of the energy released in Complex III reaction is used to move 4 H+ across
the inner membrane from the matrix to the intermembrane space.

Complex IV: Cytochrome c Oxidase

Complex IV receives the electrons from cytochrome c and it contains 13 subunits including 2
cytochromes. The electron movement flows from cyt c (carrying electrons from complex III) to
cyt a to cyt a3 as shown by the diagram below:

Note that the two cytochromes present in Complex IV (cytochromes a and a3) differ from
previously encountered cytochromes in that each has a copper atom associated with it in
addition to its iron center. The copper atom sites participate in the electron transfer process as
do the iron atom sites, with the copper atoms going back and forth between the reduced cuprous
ion (Cu+) state and the oxidized cupric ion (Cu2+) state.

In the final step of electron transfer, the electrons from cyt c and hydrogen ions from cellular
solution combine with oxygen (O2) to form water.
O2 + 4 H+ + 4 e- à 2 H2O

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It is estimated that 95% of the oxygen used by cells serves as the final electron acceptor for the
ETC. The water molecule formed in this way is released into the matrix.

During this process, 2 H+ ions are pumped out of the matrix and into the membrane space. The
energy driving this process is derived from the energy of water formation.

Inhibitors of the Electron Transport Chain

1. Rotenone, a common insecticide, is an inhibitor of Complex I. It is obtained from the
roots of several plant species. Natives in certain parts of the world have made a practice
of beating the roots and trees along riverbanks to release rotenone into the water, where
it paralyzes fishes and make them easy preys. Amytal and other barbiturates and the
widely prescribed painkiller Demerol also inhibit Complex I. All of these inhibitors inhibit
reduction of CoQ and the oxidation of FeSP.
2. Cyanide and carbon monoxide inhibit Complex IV (cytochrome c oxidase). Cyanide
binds strongly to the Fe3+ form of cytochrome a3, whereas carbon monoxide binds only
to the Fe2+ form. The inhibitory action of cyanide at this site is very potent, whereas the
principal toxicity of carbon monoxide arises from its affinity for the iron hemoglobin.
Herein lies an important distinction between the poisonous effects of cyanide and carbon
monoxide. Because animals, including humans, carry many hemoglobin molecules, they
must inhale large quantity of carbon monoxide to die from it. The same organisms,
however, possess comparatively few molecules of cytochrome a3. Consequently, a limited
exposure to cyanide can be lethal. The sudden action of cyanide attests to the organism’s
constant and immediate need for energy supplied by the ETC.

Oxidative Phosphorylation
Oxidative phosphorylation is the biochemical process by which ATP is synthesized from ADP
as a result of the transfer of electrons and hydrogen ions from NADH or FADH2 to O2 through
the electron carriers involved in the electron transport chain.

How do electron and H+ transports produce the chemical energy of ATP?

In 1961, Peter Mitchell proposed an answer to this. This was known as the chemiosmotic theory,
which states that the energy in the electron transport chain creates a proton gradient due to the
movement of protons (H+ ions) across the inner mitochondrial membrane. In this case, there is
a higher concentration of H+ in the intermembrane space than inside the matrix of the
mitochondrion.

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How are protons (H+ ions) pumped through the mitochondrial inner membrane?
Recall, three of the four protein complexes involved in the ETC chain (I, III, and IV) have a second
function besides electron transfer down the chain. They also serve as “proton pumps,”
transferring protons from the matrix side of the inner mitochondrial membrane to the
intermembrane space. Some of the H+ ions crossing the inner mitochondrial membrane come
from the reduced electron carriers, and some come from the matrix. (The details of how the H+
ions cross the inner mitochondrial membrane are not fully understood.)

Experimental evidence suggests that for every two electrons passed through the ETC:
• four (4) protons cross the inner mitochondrial membrane through complex I,
• four (4) through complex III, and
• two (2) through complex IV.

The relationship between number of protons crossing the mitochondrial inner membrane into the
intermembrane space and the electron carriers NADH and entering the electron transport chain
is as follows:
1. The oxidation of an NADH molecule results in 10 H+ ions crossing the membrane.
2. The oxidation of an FADH2 molecule results in 6 H+ ions crossing the membrane.

The difference in proton pumping results for the two oxidations stems from FADH2 initially
interacting with complex II, thus bypassing complex I which is a proton-pumping station.

How does this proton gradient form ATP?

Proton-
translocating
ATP synthase

Due to the proton pumps, there is more electrons in the intermembrane space than in the
matrix. Based on the principle of diffusion, it can be said that the protons will have the tendency
to go back to the matrix where there is less of them. But the protons cannot simply cross the inner

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membrane. They are propelled back to the matrix through a complex known as proton-
translocating ATP synthase. This compound is located on the inner membrane of the
mitochondrion and is the active enzyme that catalyzes the conversion of ADP and inorganic
phosphate to ATP. Let us talk about first the structure of this complex to understand how the flow
of electrons back to the matrix leads to production of ATP.

The proton-translocating ATP synthase (or simply ATP synthase) is a complex “rotor
engine” made of 16 different proteins.
• The complex contains the "stator," whose function is not directly involved in ATP synthesis
but is simply used to stabilize the complex as a whole.
• The F0 sector, which is embedded in the membrane, contains the proton channel where
the protons from the intermembrane space reenters the matrix. This channel rotates every
time protons pass.
• This rotation is transmitted to a “rotor” in the F1 sector. The F1 is surrounded by catalytic
unit that synthesizes the ATP. The catalytic unit converts the mechanical energy of the
rotor into chemical energy by converting ADP and phosphate group to ATP molecule.

In other words, as protons reenter the matrix through the ATP synthase, they cause for the F0 to
rotate. The rotation provides energy in the rotor of the F1 section converting ADP to ATP.

This reaction in the ATP synthase is considered a coupled reaction. Coupled reactions are pairs
of biochemical reactions that occur concurrently in which energy released by one reaction is used
in the other reaction. Oxidative phosphorylation and the oxidation reactions of the electron
transport chain are coupled systems.

Inhibitor of the ATP Synthase

Oligomycin is a polyketide antibiotic that can inhibit the ATP Synthase by binding to the F0 unit.
Oligomycin also blocks the movement of protons through F0.

Transfer of ATP Outside the Matrix

The ATP produced through oxidative phosphorylation must be moved from the matrix back to the
intermembrane space before it can be used in metabolic reactions. This is accomplished through
a transport protein known as ATP-ADP translocase embedded in the inner mitochondrial
membrane. This transport process involves movement of molecules in both directions through the
protein. For each ATP molecule transferred from the matrix to the intermembrane space an ADP,
a Pi, and an H+ ion move in the opposite direction.

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P/O Ratio for Oxidative Phosphorylation

The P/O ratio is the number of molecules ATP formed in oxidative phosphorylation per two
electrons flowing through a defined segment of the ETC. In spite of the intense study of this ratio,
its actual value remains a matter of contention.

Recent study elucidated that there is 1 ATP molecule produced in every 4 H+ ions transported
across the inner membrane of the mitochondrion. Using this fact, we can calculate the P/O of
NADH and FADH2 since we already know the amount of H+ ions they can transport during ETC:

𝑃 1 𝐴𝑇𝑃
𝑓𝑜𝑟 𝑁𝐴𝐷𝐻 = , 0 (10 𝐻! 𝑝𝑒𝑟 2 𝑒 " ) = 2.5 𝐴𝑇𝑃 𝑝𝑒𝑟 2 𝑒 "
𝑂 4 𝐻!
𝑃 1 𝐴𝑇𝑃
𝑓𝑜𝑟 𝐹𝐴𝐷𝐻# = , 0 (6 𝐻! 𝑝𝑒𝑟 2 𝑒 " ) = 1.5 𝐴𝑇𝑃 𝑝𝑒𝑟 2 𝑒 "
𝑂 4 𝐻!

The value indicates that for every NADH molecule that will enter the ETC, an equivalent of 2.5
ATP is produced during oxidative phosphorylation; and for every FADH2, 1.5 ATP is produced.
Some books prefer rounding this values off to 3 and 2, respectively. We will use the 2.5 and 1.5
values during ATP calculation as this is the most accepted value.

ATP Production for The Common Metabolic Pathway

The amount of ATP that is produced from the common metabolic pathway (Krebs Cycle, ETC,
and Oxidative Phosphorylation) can be traced using the P/O values obtained above. It is
summarized in the table below:
Pathway By-product ATP Yield
Citric Acid Cycle
- Oxidation of isocitrate to 1 NADH 2.5
α-ketoglutarate (Step 3)
- Oxidation of α- 1 NADH 2.5
ketoglutarate to succinyl
coA (Step 4)
- Cleavage of thioester 1 GTP 1
bond of succinyl coA to
form succinate
- Oxidation of succinate 1 FADH2 1.5
to fumarate
- Oxidation of L-malate to 1 NADH 2.5
oxaloacetate
TOTAL 10 ATPs

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III. METABOLISM OF CARBOHYDRATES, LIPIDS, AND PROTEINS

A. Glycolysis

Carbohydrates are the primary source of energy for humans. Thus, carbohydrate metabolism
is very related to energy production in cells. The molecule glucose is the focal point of
carbohydrate metabolism. Commonly called blood sugar, glucose is supplied to the body via the
circulatory system and, after being absorbed by a cell, can be either oxidized to yield energy
or stored as glycogen for future use. When sufficient oxygen is present (anaerobic), glucose is
totally oxidized to CO2 and H2O. However, in the absence of sufficient oxygen (anaerobic),
glucose is only partially oxidized to lactic acid. Besides supplying energy needs, glucose and
other six-carbon sugars can be converted into a variety of different sugars needed for
biosynthesis.

Glycolysis is the first stage in carbohydrate metabolism where it is oxidized to yield energy. In
this process, glucose (a 6C molecule) is converted into two molecules of pyruvate (a 3C
molecule), through a series of 10 reactions in a linear pathway.
• By-products: There is a concomitant production of ATP and NADH.
• Location: This pathway works in most cells and is characterized as anaerobic pathway
which requires no molecular oxygen. This specifically occurs in the cytoplasm of the cell,
in comparison to the Krebs Cycle and the Electron Transport Chain which occur in the
mitochondrion.

As a historical note, this is the first metabolic pathway to be elucidated by the works of many
biochemists, such as Gustav Embden, Otto Meyerhof, Carl Neuberg, Jacob Parnas, Otto
Warburg, Gerty Cori, and Carl Cori. The sequence of the reaction in glycolysis is often referred to
as the Emden-Meyerhof-Parnas pathway.

Why is glycolysis important?

For some tissues (such as brain, kidney medulla, and rapidly contracting muscles) and form some
cells (such as erythrocytes and sperm cells), glucose is the only source of metabolic energy.

Reactions of Glycolysis
Microorganisms, plants, and animals (including humans) carry out the 10 reactions of glycolysis
in similar fashion, although the rates of the individual reactions and the means by which they are
regulated differ from species to species.

In most cases, glycolysis consists of 2 phases – first phase (energy investment phase) and
second phase (energy recovery phase).
• In the first phase, a series of five reactions, glucose is broken down to two molecules of
glyceraldehyde-3-phosphate.
• In the second phase, five subsequent reactions convert these two molecules of
glyceraldehyde-3-phosphate into two molecules of pyruvate.

Phase 1 consumes 2 molecules of ATP. The later stages of glycolysis result in the production of
4 molecules of ATP. The net is 4 – 2 = 2 molecules of ATP produced per molecule of
glucose. The figure on the next page illustrates the glycolytic pathway:

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Energy Investment Phase of Glycolysis

In the first phase, glucose will be phosphorylated in C1 and C6, and the six carbon of glucose
will be cleaved to yield two 3-carbon molecules of glyceraldehyde-3-phosphate.

Reaction 1: Phosphorylation of Glucose at C6

The first reaction in glycolysis is the phosphorylation of glucose at C6 forming glucose-6-
phosphate. This is known as the first priming reaction because this is the first reaction that
consumes ATP.

Importance of this Reaction:

• Phosphorylation keeps glucose substrate in the cells. Glucose is neutral molecule and
could diffuse across the cell membrane, but phosphorylation confers a negative
charge on glucose and plasma membrane is impermeable to glucose-6-phosphate.
• Moreover, rapid conversion of glucose to glucose-6-phosphate keeps the intracellular
concentration of glucose low, favoring facilitated diffusion of glucose into the cell.
• The product glucose-6-phosphate is branched point in glucose metabolism as it is
common to several metabolic pathways.

This reaction carried out by the enzyme hexokinase or glucokinase, which are isozymes. Both
forms require Mg2+ ion cofactor. The table below shows differences between hexokinase and
glucokinase:
Hexokinase Glucokinase
- Present in almost all tissues - Present in the liver
- Non-specific enzyme (catalyzes - Highly specific to glucose
phosphorylation of other hexoses,
such as mannose and fructose)
- Can operate at normal blood glucose - Can only work when there is high level
level (4 mM) of glucose (e.g. after consuming large
amounts of carbohydrates)
- Allosterically inhibited by glucose-6- - Not allosterically inhibited
phosphate
When glucose levels are low, hexokinase is primarily responsible for phosphorylating glucose.
However, when glucose levels are high, glucose is converted by glucokinase to glucose-6-
phopshate and is eventually stored as glycogen in the liver.

Medical Note:
• The amount of glucokinase in the liver is controlled by insulin. Patients with diabetes
mellitus produce insufficient insulin. They have low levels of glucokinase, cannot tolerate
high levels of blood glucose, and produce little liver glycogen.

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Regulation: The enzyme hexokinase is one of\ the 3 regulated enzymes in glycolysis. High
levels of glucose-6-phosphate inhibit hexokinase activity until consumption by glycolysis lowers
its concentration. Thus, hexokinase reaction is one of the 3 reactions in the glycolytic pathway
that are regulated.

Reaction 2: Isomerization of Glucose-6-Phosphate to Fructose-6-Phosphate

The second step in glycolysis is an isomerization of a sugar, resulting to conversion of an
aldose (glucose-6phosphate) to a ketose (fructose-6-phosphate).

Importance of this reaction:

• The next step in glycolysis is phosphorylation at C-1, and the hemiacetal -OH of glucose,
would be more difficult to phosphorylate than a simple primary hydroxyl.
• The isomerization to fructose (with a carbonyl group at position 2 in the linear form)
activates C-3, facilitating C-C bond cleavage in the fourth step of glycolysis.

The enzyme responsible for this isomerization is phosphoglucoisomerase, also known as

glucose phosphate isomerase. In humans, the enzyme requires Mg2+ ion cofactor.

Reaction 3: Phosphorylation of Fructose-6-Phosphate at C1

The third reaction phosphorylates the C1 of fructose 6-phosphate forming fructose-1,6-
bisphophate in the presence of the enzyme phosphofructokinase. This reaction is known as
the second priming reaction as this is the second and the last reaction of glycolysis that uses
ATP.

Importance of This Reaction:

• This reaction commits the cell in metabolizing glucose rather than converting it to
another sugar or storing it. After fructose-1,6-bisphosphate is formed from the original
sugar, not other pathways are available, and molecules must undergo the remaining
reactions of glycolysis.

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Regulation: The enzyme phosphofructokinase is also one of the 3 regulated enzymes in

glycolysis.
• The first regulator of this enzyme is ATP. High levels of ATP decrease the activity of this
enzyme, and low levels of ATP stimulates the reaction.
• Citrate, an intermediate in the citric acid cycle, is also an allosteric inhibitor of
phosphofructokinase. Thus, glycolysis and the citric acid cycle are coupled via
phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which
“feeds” the citric acid cycle under aerobic conditions) slows down.

Reaction 4: Cleavage of Fructose-1,6-Bisphosphate Forming 3-Carbon Intermediates

The enzyme aldolase cleaves fructose-1,6-bisphosphate to yield two triose phosphates,
dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate.

Reaction 5: Isomerization of DHAP to Glyceraldehyde-3-Phosphate

In this reaction, dihydroxyacetone phosphate is converted to glyceraldehyde-3-phosphate by
the enzyme triose phosphate isomerase.

Importance of this reaction:

• Of the two products of the aldolase reaction, only glyceraldehyde-3-phosphate goes
directly into the second phase of glycolysis since most of the remaining steps are oxidative
in nature. DHAP has a ketone group which cannot be oxidized easily, and must, therefore,
be converted to glyceraldehyde-3-phosphate. In other words, this reaction permits both
products of the aldolase reaction to continue in the glycolytic pathway.

The enzyme triose phosphate isomerase is one of the enzymes that have evolved to a state of
catalytic perfection. So, it can be said, that 2 glyceraldehyde-3-phosphate molecules proceed
to the remaining reactions of glycolysis.

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Energy Recovery Phase

The second phase of the glycolytic pathway involves the reactions that convert the metabolic
energy in the glucose molecule into ATP. Altogether, 4 new ATP molecules are produced. The 2
ATP produced in this phase offset the 2 ATP consumed in the first phase, resulting to a net
production of 2 ATP.

Reaction 6: Oxidation and Phosphorylation of Glyceraldehyde-3-Phosphate

In this step, glyceraldehyde-3phosphate is oxidized to 1,3-bisphosphoglycerate by
glyceraldehyde-3-phosphate dehydrogenase. Note, that the phosphate group used in the
phosphorylation of the substrate is not coming from ATP, but from inorganic phosphate.

The newly added phosphate group in 1,3-bisphosphoglycerate is a high-energy phosphate

group. A high-energy phosphate group is produced when a phosphate group is attached to a
carbon atom that is also participating in a carbon–carbon or carbon–oxygen double bond.

Inhibitor: The enzyme involved in this reaction can be inactivated by:

• Iodoacetate – reacts with and blocks the essential cysteine residue in the active site.
• Arsenate (AsO43+) – a competitive inhibitor of the enzyme since it is analogous to
phosphate (PO43+). Arsenate is an effective substrate in this reaction, forming 1-arseno-
3-phosphoglycerate, but acyl arsenates are quite unstable and are rapidly hydrolyzed to
yield 3-phosphoglycerate, the product of the seventh reaction of glycolysis. The result is
that glycolysis continues in the presence of arsenate, but the molecule of ATP formed in
reaction 7 (phosphoglycerate kinase) is not made because this step has been bypassed.

Reaction 7: Formation of 3-Phosphoglycerate and First 2 ATP Molecules

In this step, the enzyme phosphoglycerate kinase (also known as phosphoglycerokinase)
transfers a phosphate group from 1,3-bisphosphoglycerate to ADP to form an ATP and 3-
phosphoglycerate. The enzyme of this reaction requires Mg2+ ion cofactor.

Importance of this reaction:

• Because each glucose molecule sends 2 molecules of glyceraldehyde-3-phosphate
into the second phase of glycolysis, 2 ATP molecules are produced in this step. And,
because two ATPs were consumed per glucose in the first-phase reactions, this reaction

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only “pays off” the ATP debt created by the priming reactions. This is the reason why this
step is the break-even step of the glycolytic pathway.

ATP production in this step involves substrate-level phosphorylation, wherein ATP is produced
is not from oxidative phosphorylation.

Reaction 8: Isomerization of 3-Phosphoglycerate to 2-Phosphoglycerate

In this step, the enzyme phosphoglycerate mutase (also known as phosphoglyceromutase)
transfers the phosphate group of 3-phosphoglycerate from C3 to C2 forming 2-
phosphoglycerate. (The term mutase is applied to enzymes that catalyze migration of a
functional group within a substrate molecule.)

Reaction 9: Dehydration of 2-Phosphoglycerate Forming Phosphoenolpyruvate (PEP)

In this step, the enzyme enolase catalyzes the removal of water molecule from 2-
phosphoglycerate forming phosphoenolpyruvate. Enolase requires a Mg2+ ion cofactor.

Importance of this reaction:

• This converts the phosphate group in PEP into a high-energy phosphate group as it is
already attached to carbon involved in C-C double bond.

Inhibitor: The enzyme is strongly inhibited by fluoride ion in the presence of phosphate.

Reaction 10: Formation of Pyruvate and Final 2 ATP Molecules

The second ATP-synthesizing reaction of glycolysis is catalyzed by pyruvate kinase, which
brings the pathway at last to its final product, pyruvate. Pyruvate kinase mediates the transfer of
a phosphate group from phosphoenolpyruvate to ADP to make ATP and pyruvate. The
reaction requires Mg2+ ion and is stimulated by K+ and certain other monovalent cations.

Again, because each glucose molecule sends two molecules of glyceraldehyde-3-phosphate into
the second phase of glycolysis, 2 ATP molecules are produced in this step.

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Regulation: Pyruvate kinase is the last of the 3 enzymes that are regulated in glycolysis. It is
activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl coA, and
alanine. When this enzyme is inhibited, the PEP will be used as a substrate for glucose synthesis
in the process known as gluconeogenesis.

Summary of the Reactions of Glycolysis

The net overall equation for the process of glycolysis is:

Regulation of Glycolysis

In glycolysis, 3 reactions are the control points:

• Reaction 1, the conversion of glucose to glucose 6-phosphate, involves the enzyme
hexokinase. This enzyme is inhibited by glucose 6-phosphate, the substance produced
by its action (feedback inhibition).
• Reaction 3, where fructose 6-phosphate is converted to fructose 1,6-bisphosphate by the
enzyme phosphofructokinase, ATP and citrate inhibit enzyme activity. A high ATP
concentration, which is characteristic of a state of low energy consumption, thus stops
glycolysis at the fructose 6-phosphate stage.
• Reaction 10, the conversion of phosphoenolpyruvate to pyruvate, is the last control point.
Pyruvate kinase, the enzyme needed at this point, is inhibited by high ATP concentrations.

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Fates of Pyruvate
The product of glycolysis, pyruvate, is a versatile metabolite than can be used in several ways.
The figure below shows the different fates of pyruvate:

• In some tissues where oxygen is plentiful (aerobic), pyruvate is converted into an acetyl
group of an acetyl coA molecule. As we know, acetyl coA is metabolized into the citric
acid cycle where the acetyl portion is completely oxidized to CO2 molecules.
• Alternatively, in tissues where there is low supply of oxygen (anaerobic), pyruvate can
undergo reduction to form lactate with concomitant oxidation of NADH to NAD+. This
process is termed as lactate fermentation.
• In microorganisms such as yeast, and in some plant tissues, pyruvate can be reduced
into ethanol also with concomitant oxidation of NADH to NAD+. This process is termed
as alcoholic fermentation.

Entry of Carbohydrate Metabolite (Pyruvate) to Citric Acid Cycle

As mentioned earlier, pyruvate produced by glycolysis is a significant source of acetyl coA for
the citric acid cycle. Because, in eukaryotic cells, glycolysis occurs in the cytoplasm, whereas
the citric acid cycle, ETC and oxidative phosphorylation take place in the mitochondria,
pyruvate must first enter the mitochondria to enter the citric cycle.

The oxidative decarboxylation of pyruvate to acetyl-CoA is the connecting link between

glycolysis and the citric acid cycle. The reaction is catalyzed by pyruvate dehydrogenase, a
multienzyme complex. This reaction can be summarized by the equation below:

The enzyme complex involved contains three different enzymes, each with numerous subunits.
The overall reaction process involves four separate steps and requires NAD+, CoA, FAD, and
two other coenzymes (lipoic acid and thiamine pyrophosphate (TPP)).

Medical Note: Alcoholism causes TPP deficiency, which affects the function of pyruvate
dehydrogenase. In this case, pyruvate will have a higher chance of being converted to lactate
which can accumulate in the tissues and cause lactic acidosis.

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The Fates of Cytosolic NADH Produced in Glycolysis

Most of the NADH used in electron transport is produced in the mitochondrial matrix, an
appropriate site because NADH is oxidized by Complex I on the matrix side of the inner
membrane. Furthermore, the inner mitochondrial membrane is impermeable to NADH.

Recall, however, that NADH is produced in glycolysis by glyceraldehyde-3-phosphate

dehydrogenase in the cytoplasm. Eukaryotic cells have several shuttle systems that harvest
the electrons of cytosolic NADH for delivery to mitochondria without transporting NADH across
the inner membrane.

1. Glycerol-3-Phosphate Shuttle
In the glycerol-3-phosphate shuttle, two different glycerophosphate dehydrogenases,
one in the cytoplasm and one on the outer face of the mitochondrial inner membrane,
work together to carry electrons into the mitochondrial matrix. NADH produced in the
cytoplasm transfers its electrons to dihydroxyacetone phosphate, thus reducing it to
glycerol-3-phosphate. This metabolite is reoxidized by the FAD-dependent mitochondrial
membrane enzyme to reform dihydroxyacetone phosphate and enzyme-bound FADH2.
Thus, via this shuttle, cytosolic NADH can be used to produce mitochondrial FADH2. As a
result, cytosolic NADH oxidized via this shuttle route yields only 1.5 molecules of ATP. This
shuttle operates in the muscles and nerve cells.

2. Malate-Aspartate Shuttle
The second electron shuttle system, called the malate–aspartate shuttle, is shown in figure
below. Oxaloacetate is reduced to malate in the cytoplasm, acquiring the electrons of NADH
(which is oxidized to NAD+). Malate is transported across the inner membrane, where it is
reoxidized by malate dehydrogenase to oxaloacetate, converting NAD+ to NADH in the
matrix. This mitochondrial NADH readily enters the electron-transport chain. The
oxaloacetate produced in this reaction cannot cross the inner membrane and must be
transaminated to form aspartate, which can be transported across the membrane to the
cytosolic side. Transamination in the cytoplasm recycles aspartate back to oxaloacetate. In
contrast to the glycerol phosphate shuttle, this shuttle produces NADH in the matrix, the full
2.5 ATPs per NADH are recovered. This shuttle operates in the heart cells.

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Net Yield of ATP for A Molecule of Glucose

• Aerobic Metabolism of Glucose

Aerobic metabolism of glucose in eukaryotic cells involved the combined pathways of
glycolysis, Krebs cycle, electron transport, and oxidative phosphorylation. Overall, this
process yields a net of approximately 30-32 molecules of ATP per molecule of glucose,
depending on the shuttle system used. The table below summarizes the ATP production
of aerobic glucose catabolism.

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• Anaerobic Metabolism of Glucose

Anaerobic metabolism of glucose in eukaryotic cells converts the 2 pyruvate molecules
produced in glycolysis to 2 molecules lactate. This process requires 2 NADH molecules.
Thus, its energy yield is just the 2 net ATP molecules produced in the glycolytic pathway.
The NADH produced in the pathway is not converted to ATP in the mitochondrion since it
is used in the conversion of pyruvate to lactate.

B. Other Metabolic Pathways of Carbohydrates

Aside from glycolysis, carbohydrates also undergo other pathways a illustrated below:

I. Gluconeogenesis
• Definition: Gluconeogenesis is the metabolic pathway by which glucose is
synthesized from non-carbohydrate materials, such as pyruvate, lactate (from
hard-working muscles and from red blood cells), glycerol (from triacylglycerol
hydrolysis), and certain amino acids (from dietary protein hydrolysis or from muscle
protein during starvation). This occurs when the blood glucose level is incredibly low.
• Location: About 90% of gluconeogenesis takes place in the liver, and to some extent
in the kidneys. Its cellular location is partly mitochondrial and partly cytoplasmic.
• Importance:
1. This pathway covers up the glucose demands in the body when glucose is not
consumed in the diet.
2. Furthermore, working muscles are anaerobic and produce large amounts of
pyruvate which is converted to lactate. Gluconeogenesis salvages this
pyruvate and lactate and reconverts it to glucose.
• Cori Cycle
As mentioned, lactate can also be a non-carbohydrate precursor in the production of
glucose through gluconeogenesis. Lactate is formed in the muscles during strenuous
exercise due to the low supply of oxygen. The lactate so produced diffuses from
muscle cells into the blood, where it is transported to the liver. Here the enzyme lactate
dehydrogenase (the same enzyme that catalyzes lactate formation in muscle)

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converts lactate back to pyruvate. The newly formed pyruvate is then converted via
gluconeogenesis to glucose, which enters the bloodstream and goes to the muscles.
This cyclic process is called the Cori cycle. It is a cyclic biochemical process in which
glucose is converted to lactate in muscle tissue, the lactate is reconverted to glucose
in the liver, and the glucose is returned to the muscle tissue. The figure below
summarizes the Cori cycle.

II. Glycogen Metabolism

Glycogen, a branched polymeric form of glucose, is the storage form of carbohydrates in
humans and animals. It is found primarily in muscle and liver tissue. In muscles, it is the
source of glucose needed for glycolysis. In the liver, it is the source of glucose needed to
maintain normal glucose levels in the blood.

Glycogen is synthesized whenever the body has excess glucose (glycogenesis). At the same
time, when supply of glucose is low, it can also be degraded to obtain energy
(glycogenolysis).

Glycogenesis
• Definition: When our body does need ATP, the glucose 6-phosphate produced in
the first step of glycolysis will undergo different set of reaction to synthesize glycogen.
This metabolic pathway is known as glycogenesis. This is a polymerization
pathway in which glucose subunits are connected to form long chains.
• Location: This occurs primarily in the liver, and to some extent in the muscles.
• Importance: This occurs when there is a high level of glucose in the body (e.g., after
eating high levels of carbohydrates).
• This process consumes a total of 2 ATP, which is reasonable as this is an anabolic
pathway.

Glycogenolysis
• Definition: When our body needs ATP and there’s no more glucose available in our
blood system, our body will get our stored glycogen and convert it to glucose 6-
phosphate, which will proceed to glycolysis. This process is called glycogenolysis.
• Location: This occurs primarily in the muscles and in the brain.

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• When glycogen rather than free glucose is the starting material for glycolysis, the net
gain in ATP is 3 molecules rather than 2 for each glucose processed. Glucose from
glycogen enters glycolysis at Step 2, as glucose 6-phosphate, and thus bypasses
ATP-consuming Step 1. Thus, glycogen is a more effective energy source than is
free glucose.

III. Pentose Phosphate Pathway

• Definition: The pentose phosphate
pathway is the metabolic pathway by
which glucose 6-phosphate is used to
produce ribose 5-phosphate (a
pentose phosphate) and numerous
other sugar phosphates. It has
concomitant production of
nicotinamide adenine dinucleotide
phosphate (NADPH) whose structure
i shown at the side.
• Location: The operation of the
pentose phosphate pathway is
significant in cells that produce lipids:
fatty tissue, liver, mammary glands,
and the adrenal cortex (an active
producer of steroid lipids).
• Importance:
1. synthesis of the reduced form of
coenzyme nicotinamide adenine
dinucleotide phosphate
(NADPH) needed in lipid
biosynthesis; and
2. production of ribose 5-phosphate, a pentose derivative needed for the
synthesis of nucleic acids and many coenzymes.

IV. Hormonal Controls of Carbohydrate Metabolism

1. Insulin is produced by the beta cells of the pancreas. It promotes the uptake
and utilization of glucose by the cells. Thus, in effect lowering down glucose
levels in the blood. It is also involved in lipid metabolism. Insulin is released
when there’s high blood glucose levels and binds to the protein receptors that
serves as an entry point of glucose to the cells. It increases the rate of
glycogenesis, glycolysis, and fatty acid synthesis.
2. Glucagon is produced by the alpha cells of the pancreas. It is released at
when theirs low blood sugar. Its function is to speed up glycogenolysis and
gluconeogenesis in the liver.
3. Epinephrine or adrenaline is released by adrenal glands in response of fear,
anger, or excitement. It has similar effects as glucagon but targeting muscle
cells for quick action. It is also associated to lipid metabolism.

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C. Storage and Mobilization of Fats

Most cells in the body have limited capability for storage of

TAGs. However, this activity is the major function of
specialized cells called adipocytes, found in adipose tissue.
An adipocyte is a triacylglycerol-storing cell. Adipose tissue
is tissue that contains large numbers of adipocyte cells. The
figure below is an illustration of an adipocyte.

Adipose tissue is located primarily directly beneath the skin

(subcutaneous), particularly in the abdominal region, and in
areas around vital organs. Besides its function as a storage
location for the chemical energy inherent in TAGs,
subcutaneous adipose tissue serves as an insulator against
excessive heat loss to the environment and provides organs
with protection against physical shock.

Adipose cells are among the largest cells in the body. They differ from other cells in that most of
the cytoplasm has been replaced with a large triacylglycerol droplet. This droplet accounts for
nearly the entire volume of the cell. As newly formed TAGs are imported into an adipose cell, they
form small droplets at the periphery of the cell that later merge with the large central droplet.

How does our body use TAG for energy production?

Use of the TAGs stored in adipose tissue for energy production is triggered by several hormones,
including epinephrine and glucagon. Hormonal interaction with adipose cell membrane
receptors stimulates production of cyclic adenosine monophosphate (cAMP) from ATP inside the
adipose cell. In a series of enzymatic reactions, the cAMP activates hormone-sensitive lipase
(HSL) through phosphorylation. HSL is the lipase needed for triacylglycerol hydrolysis, a
prerequisite for fatty acids to enter the bloodstream from an adipose cell. This cAMP activation
process is illustrated below.

The overall process of tapping the body’s triacylglycerol energy reserves (adipose tissue) for
energy is called triacylglycerol mobilization. At the end of this process, 3 fatty acid molecules
and 1 glycerol molecule are liberated for every molecule of triacylglycerol.

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D. Catabolism of Glycerol

During triacylglycerol mobilization, one molecule of glycerol is produced for each triacylglycerol
molecule completely hydrolyzed. After entering the bloodstream, the glycerol travels to the liver
or kidneys, where the first stage of glycerol metabolism occurs. At these locations it is converted
to dihydroxyacetone phosphate, a glycolysis intermediate, in a 2-step process.

The first step involves phosphorylation of a primary hydroxyl group of the glycerol. In the
second step, glycerol’s secondary alcohol group is oxidized to a ketone.

The dihydroxyacetone phosphate product can be converted to pyruvate, then acetyl CoA, and
finally carbon dioxide. Dihydroxyacetone phosphate formation from glycerol represents the first
of several situations that will be encountered where lipid and carbohydrate metabolism are
connected.

E. b-Oxidation of Fatty Acids

There are three parts to the process by which fatty acids are broken down to obtain energy:
1. The fatty acid must be activated by bonding to coenzyme A.
2. The fatty acid must be transported into the mitochondrial matrix by a shuttle mechanism.
3. The fatty acid must be repeatedly oxidized, cycling through a series of four reactions, to
produce acetyl CoA, FADH2, and NADH.

Fatty Acid Activation

In the first stage of fatty acid catabolism, the fatty acid is converted to a high-energy derivative of
coenzyme A. Reactants are the fatty acid, coenzyme A, and a molecule of ATP.

• Location: The first stage of fatty acid catabolism its activation which occurs in the outer
mitochondrial membrane.
• This reaction requires the expenditure of two high-energy phosphate bonds from a
single ATP molecule; the ATP is converted to AMP rather than ADP. This is energetically
equivalent to consuming 2 ATP molecules.

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The activated fatty acid–CoA molecule is called acyl CoA. The difference between the
designations acyl CoA and acetyl CoA is that acyl refers to a random-length fatty acid carbon
chain that is covalently bonded to coenzyme A, whereas acetyl refers to a two-carbon chain
covalently bonded to coenzyme A.

Transport of Acyl coA Across Inner Mitochondrial Membrane

Acyl coA is too large to pass through the inner mitochondrial membrane to the mitochondrial
matrix, where the enzymes needed for fatty acid oxidation are located. A shuttle mechanism
involving the molecule L-carnitine effects the entry of acyl CoA into the matrix.

Carnitine acyltransferase I, associated with the outer mitochondrial membrane, catalyzes the
formation of the O-acylcarnitine, which is then transported across the inner membrane by a
translocase. At this point, the acylcarnitine is passed to carnitine acyltransferase II on the
matrix side of the inner membrane, which transfers the fatty acyl group back to CoA to re-form
the acyl coA, leaving free carnitine, which can return across the membrane via the translocase.

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β-Oxidation
• Definition: The acyl coA then undergoes a sequence of reactions repeatedly cleaves two
carbon units (acetyl) from the carboxyl end of molecule. This repetitive four-reaction
sequence is called the β-oxidation pathway because the second carbon from the
carboxyl end of the chain, the β carbon, is the carbon atom that is oxidized.

• Location: Mitochondrial matrix

For saturated fatty acids, the process of β-oxidation is a repetitive series of four biochemical
reactions that degrade acyl CoA to acetyl CoA by removing two carbon atoms at a time, with
concomitant production of FADH2 and NADH. Each repetition of the four-reaction sequence
generates an acetyl CoA molecule and an acyl CoA molecule that has two fewer carbon
atoms. This pathway involves the following functional group changes at the β-carbon and the
following reaction types.

The overall strategy in the first three steps is to create a carbonyl group on the β-carbon by
oxidizing the Cα–Cβ bond to form a Cα=Cβ bond, with subsequent hydration and oxidation. In
essence, this cycle is directly analogous to the sequence of reactions converting succinate to
oxaloacetate in the citric acid cycle. The fourth reaction of the cycle cleaves the β-keto ester in
a reverse Claisen condensation. The following are the details of the four reactions.

Step 1: First Dehydrogenation

Hydrogen atoms are removed from the α and β carbons, creating a double bond between
these two carbon atoms. FAD is the oxidizing agent, and an FADH2 molecule is a product.

Step 2: Hydration
A molecule of water is added across the trans double bond, producing a secondary alcohol at
the β-carbon position. Again, the enzyme involved is stereospecific in that only the L-hydroxy
isomer is produced from the trans double bond.

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Step 3: Second Dehydrogenation

Removal of two hydrogen atoms converts the β-hydroxy group to a keto group, with NAD+
serving as the oxidizing agent. NADH molecule is produced. The required enzyme exhibits
absolute stereospecificity for the L-isomer.

Step 4: Thiolysis
The fatty acid carbon chain is broken between the α and β carbons by reaction with a coenzyme
A molecule. The result is an acetyl CoA molecule and a new acyl CoA molecule that is shorter
by two carbon atoms than its predecessor.

The new acyl CoA molecule (now shorter by two carbons) is recycled through the same set of
four reactions again. This yields another acetyl CoA, a
two-carbon-shorter new acyl CoA, FADH2, and NADH.
Recycling occurs again and again, until the entire fatty
acid is converted to acetyl CoA. Thus, the fatty acid
carbon chain is sequentially degraded, two carbons at a
time.

The fatty acids normally found in dietary triacylglycerols

contain an even number of carbon atoms. Thus, the
number of acetyl CoA molecules produced in the β-
oxidation pathway is equal to half the number of
carbon atoms in the fatty acid. The number
of repetitions of the β-oxidation pathway that are
needed to produce the acetyl CoA is always one less
than the number of acetyl CoA molecules produced
because the last repetition produces two acetyl CoA
molecules as a C4 unit splits into two C2 units. Each
repetition of the cycle produces 1 FADH2 molecule and
1 NADH molecule.

The figure on the left summarizes the β-oxidation of a

stearic acid molecule. Stearic acid is a saturated fatty
acid with 18 carbons; thus it will produce a total of 9
acetyl coA upon completion of its β-oxidation pathway.
It would take 8 repetitions to complete the pathway;
thus, upon completion, 8 FADH2 molecules and 8 NADH molecules are produced.

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ATP Yield of Catabolism of Fatty Acids

Let us consider the ATP yield of stearic acid, assuming all acetyl coA produced in its β-
oxidation proceeds to the citric acid cycle and the ETC.
Reaction By-product ATP Yield
Activation
- Conversion of stearic acid to
-2
corresponding acyl coA
β-Oxidation
8 FADH2 12
- 8 repetitions
8 NADH 20
Citric Acid Cycle
- 9 cycles (9 acetyl coA are
produced in the β-oxidation 10 ATP per cycle 90
of stearic acid
TOTAL 120 ATPs

Comparison Between Energy Yield of Glucose and Fatty Acid Oxidation

Let us try to compare the energy yield for the complete oxidation of glucose and complete
oxidation of stearic acid.

Complete oxidation of glucose usually gives 30 ATP, while complete oxidation of stearic acid
gives 120 ATP. This means that a stearic acid molecule produces four times as much ATP as a
glucose molecule. Taking into account the fact that glucose has only 3 carbon atoms and stearic
acid has 18 carbon atoms still shows more ATP production from the fatty acid.
3 glucose (18C) à 90 ATP
1 stearic acid (18C) à 120 ATP

Thus, on the basis of equal numbers of carbon atoms, lipids are 33% more efficient than
carbohydrates as energy-storage systems. On an equal-mass basis, fatty acids produce 2.5
times as much energy per gram as carbohydrates (glucose); this is shown by the following
calculation involving 1 g of stearic acid and 1 g of glucose.

The fact that fatty acids (stearic acid) yield 2.5 times as much energy per gram as carbohydrates
(glucose) means that, in terms of calories consumed, the former “do 2.5 times as much damage”
to a person on a diet. On the other hand, fatty acids are much better energy-storing molecules
than is glucose; they can store more than twice as much energy per gram than glucose.

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Is the preferred fuel for “running” the human body fatty acids, which yield 2.5 times as
much energy per gram as glucose, or is it glucose?
• In a normally functioning human body, certain organs use both fuels, others prefer
glucose, and still others prefer fatty acids. Some generalizations about “fuel” use are:
1. Skeletal muscle uses glucose (from glycogen) when in an active state. In a resting
state, it uses fatty acids.
2. Cardiac muscle depends first on fatty acids and secondarily on ketone bodies,
glucose, and lactate.
3. The liver uses fatty acids as the preferred fuel.
4. Brain function is maintained by glucose and ketone bodies (Section 25-6). Fatty
acids cannot cross the blood–brain barrier and thus are unavailable.

F. Ketones Bodies

Ordinarily, when there is adequate balance between lipid and carbohydrate metabolism, most
of the acetyl CoA produced from the β-oxidation pathway is further processed through the citric
acid cycle.

The first step of the citric acid cycle involves the reaction between oxaloacetate and acetyl CoA.
Sufficient oxaloacetate must be present for the acetyl CoA to react with. Oxaloacetate
concentration depends on pyruvate produced from glycolysis, since oxaloacetate is produced
from carboxylation of pyruvate by pyruvate carboxylase.

Certain body conditions upset the lipid–carbohydrate balance required for acetyl CoA generated
by fatty acids to be processed by the citric acid cycle. These conditions include:
1. dietary intake high in fat and low in carbohydrates (ketogenic diet);
2. diabetic conditions in which the body cannot adequately process glucose even though it
is present; and
3. prolonged fasting conditions, including starvation, where glycogen supplies are
exhausted.

Under these conditions, the problem of inadequate oxaloacetate supplies arises. The acetyl coA
will build up in the body. The excess acetyl CoA is diverted to the formation of ketone bodies,
specifically acetoacetate, β-hydroxybutyrate, and acetone.

Ketogenesis is the metabolic pathway by which ketone bodies are synthesized from acetyl CoA.
The primary site for this process is liver mitochondria. There, 2 acetyl coA condenses to
produce acetoacetyl coA. Hydrolysis of acetoacetyl coA yields acetoacetate, which can be
reduced to form β-hydroxybutyrate or decarboxylated to form acetone.

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Under normal conditions, the liver sends these compounds into the bloodstream to be carried
to tissues and utilized there as a source of energy via the common catabolic pathway. The brain,
for example, normally uses glucose as an energy source. During periods of starvation, however,
ketone bodies may serve as the major energy source for the brain.

Ketosis
Under normal metabolic conditions (an appropriate glucose–fatty acid balance), the concentration
of ketone bodies in the blood is very low—about 1mg/100 mL. Abnormal metabolic conditions
produce elevated blood ketone levels, levels 50-100 times greater than normal. Excess
accumulation of ketone bodies in blood (20 mg/100 mL) is called ketonemia. At a level of 70
mg/100 mL, the renal threshold is exceeded and ketone bodies are excreted in the urine, a
condition called ketonuria.

Ketosis is the body condition in which high levels of ketone bodies are present in both the blood
and urine. Both ketonemia and ketonuria are contributors to this condition. Ketosis is often
detectable by the smell of acetone on a person’s breath; acetone is very volatile and is excreted
through the lungs. For most persons following a low-carbohydrate diet (ketogenic diet), the
effects of ketosis include headache, dry mouth, and sometimes acetone-smelling breath.
Typically, the same is true for fasting situations.

A serious to extremely serious ketosis problem called ketoacidosis can develop in persons with
uncontrolled Type 1 diabetes. Two of the three ketone bodies, acetoacetate and β-
hydroxybutyrate, are acids; a carboxyl group is present in their structure. At elevated levels, the
presence of these two ketone bodies can cause a significant decrease in blood pH. This change
in blood acidity, if left untreated, leads to heavy breathing (acidic blood carries less oxygen) and
increased urine output that can lead to dehydration. Ultimately the condition of ketoacidosis can
cause coma and death. Ketoacidosis is also called metabolic acidosis to distinguish it from
respiratory acidosis, which is not linked to ketone bodies.

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G. Amino Acid Utilization

In biochemical energy production standpoint, proteins come last in terms of contribution to the
body’s ATP production. However, despite its minor role in energy production, protein metabolism
plays an important role in maintaining good health. The amino acids obtained from proteins are
needed for both protein synthesis and synthesis of other nitrogen-containing compounds
in the cell.

In the blood stream, amino acids will be supplied to the amino acid pool, which is the total supply
of free amino acid available for use in the human body. Other sources of amino acid include
protein turnover and synthesized non-essential amino acid by the liver. The human body
continuously synthesize and degrade amino acids through disease, injury, or just wear and
tear. This process of degradation and resynthesize of proteins in our body is called protein
turnover.

For a healthy adult, the amount of nitrogen taken in from proteins must be equal to amount of
nitrogen secreted in the urine. This is what we call nitrogen balance. Imbalance can happen in
two ways: negative or positive nitrogen imbalance.

Negative nitrogen imbalance happens when the amount of nitrogen from proteins is less than
the amount excreted. This can mean a number of things like the person is experiencing tissue
wasting (proteins from tissues are experiencing fast degradation), poor diet, or starvation.
Positive nitrogen imbalance happens when the amount of nitrogen from proteins is more than
the amount excreted in the urine. This phenomenon is common to growing teenagers and
pregnant mother, wherein protein is being used for growth of both the teenager and the baby.
However, this can also happen to patients that are recovering from illness, wherein their body is
being repaired or recuperating.

Unlike lipids and carbohydrates, protein doesn’t have any special storage compound. Thus, we
need to constantly replenish the amino acid pool through our diet.

Amino acids are used in the body in a variety of ways:

1. use in protein synthesis to replenish or repair old tissue or create new ones.
2. use to synthesize non-protein nitrogen-containing compounds such as nucleic acids,
heme of hemoglobin, choline and ethanolamine of phospholipids, and
neurotransmitters like dopamine and serotonin. Note that tryptophan is used to
synthesize serotonin (happy-hormone) and tyrosine is used to create norepinephrine
(stress-hormone) and dopamine (reward-hormone). See figure below for their structural
differences.

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3. use to synthesize non-essential amino acids. The table below summarizes the products
of converting essential amino acids to non-essential amino acids and their required
metabolic precursors.

4. use as energy source by conversion to precursor chemicals used in the different metabolic
pathways.

Amino acids are further degraded into nitrogen portion and carbon portion. The fate of these two
amino acid portions are presented in the figure below. Usually the carbon portion of the amino
acid goes to energy production while the nitrogen portion is utilized for synthesis of different
nitrogen containing compounds or excreted via urea cycle.

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Catabolism of the Nitrogen Portion of Amino Acids

Unlike carbohydrates and lipids, amino acids contain nitrogen, which cannot be processed by
our body to convert to energy. Thus, the nitrogen portion of the protein must be removed from its
carbon component. Then, the carbon component can be used for energy. There are two ways in
which nitrogen can be removed from the amino acid:
(1) transamination and
(2) oxidative deamination.
The nitrogen of the products that result from these reactions are further processed through urea
cycle to be converted into urea which can be excreted through the kidneys.

The figure below combines the transamination and oxidative deamination reactions and those
of the urea cycle into a single diagram, which serves as a useful summary of important reactions
associated with the nitrogen portion of protein metabolism.

1. Transamination
• Definition: Transamination is a biochemical reaction that involves the
interchange of amino group of an α-amino acid with the keto group (C=O) of
an α-keto acid with the aid of aminotransferase enzyme and pyridoxal
phosphate (PLP), a coenzyme produced from pyridoxine (vitamin B6). This
interchange results in a new amino acid and a new keto acid being produced.
(See the general reaction of transamination on the next page.)
• Location: The liver

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• Importance: There are 20 amino acids all in all that are commonly found in
proteins. All of these amino acids will undergo transamination. However, there are
only 2 keto acids commonly encountered as reactants in transamination,
specifically α-ketoglutarate and oxaloacetate, which are both intermediates of
the citric acid cycle. This limits amino acid products to only two. With many amino
acid reactants and only two amino acid products, the purpose of transamination
becomes apparent. Its purpose is to collect the amino groups from many
different amino acids into just two amino acids, thus simplifying greatly the
process of amino acid metabolism. Only two amino acids then need to be
processed further with their nitrogen atoms ultimately being eliminated from the
body in urea.

As mentioned, there can only be 2 possible amino acid products in a

transamination reaction:

a. Transamination Resulting to Glutamate: The most important

transamination reaction in protein metabolism is the transamination
requiring α-ketoglutarate as the keto-acid, which accepts amino group
from any of the 20 amino acids to produce glutamate. The figure below
shows a transamination reaction between α-ketoglutarate and a generic
amino acid:

The glutamate can either:

(1) go perform another transamination with oxaloacetate or
(2) produce ammonium ion (NH4+) via oxidative deamination.

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b. Transamination Resulting to Aspartate

As mentioned previously, glutamate can undergo further transamination
with oxaloacetate can transfer amino groups via transamination
process. This transamination produces aspartate and regenerate α-
ketoglutarate. The “recycled” α-ketoglutarate can react with other amino
acid and produce another glutamate.

The generated aspartate in this reaction is used in the urea cycle along
with the generated ammonium ion (NH4+).

2. Oxidative Deamination
• Definition: Not all glutamates will react with oxaloacetate to produce aspartate.
Others will undergo oxidative deamination. Oxidative deamination is a
biochemical reaction in which an glutamate is converted to a α-ketoglutarate with
the release of ammonium ion (NH4+).
• Location: This reaction primarily occurs in the liver and kidney mitochondria.
• Oxidative deamination of glutamate requires the conversion of NAD+ to NADH with
the help of the enzyme glutamate dehydrogenase. Note that accumulation of
this ammonium ion is toxic to our body. Thus, it will be expelled via the urea
cycle.

3. Urea Cycle
• Definition: From a nitrogen standpoint, the net effect of transamination and
deamination reactions is production of ammonium ions and aspartate
molecules. Both of these nitrogen-carrying entities are processed further in the
urea cycle. The urea cycle is a biochemical pathway in which urea is produced
from ammonium ions and aspartate.
• Location: This process is done in the liver and urea will be transported by the
blood into the kidneys for excretion. In terms of cellular location, it is partly
mitochondrial and partly cytosolic.

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• Ammonium ions enter the cycle indirectly, being first incorporated into another
molecule (carbamoyl phosphate) that then enters the cycle’s first step.
Aspartate molecules enter the cycle directly in the cycle’s second step.
• The figure below illustrates the process in the urea cycle with their respective
location inside the cell. As illustrated, it’s partly cytosolic and partly mitochondrial.

• The urea produced in this process is excreted through the urine. In fact, urea is
the solute present in the greatest quantity in urine. Its presence has nothing to
do with the color and odor of urine, and it is odorless and colorless in solution. (The
pale yellow color of urine is due to small amounts of urobilin and related
compounds.)
• The amount of urea in urine is significantly affected by dietary intake. Large high-
protein-content meals often provide protein amounts in excess of the body’s
needs, and the excess protein cannot be stored. Processing of the nitrogen content
of the excess protein load increases the urea concentration in urine.

Fates of the Amino Acid Carbon Skeleton

After transamination and deamination, the α-ketoacid produced contains carbon skeletons form
the amino acid. Because each of the 20 amino acids has a different carbon skeleton, each amino
acid has a different degradation pathway for its carbon skeleton.

The degradation pathways of these carbon skeletons, although very different from each other,
result only to common 7 degradation products. Four of the seven degradation products are
citric acid cycle intermediates: α-ketoglutarate, succinyl CoA, fumarate, and oxaloacetate.
The other three products are pyruvate, acetyl CoA, and acetoacetyl CoA.

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The figure below relates these seven degradation products to the amino acids from which they
are obtained. Some amino acids appear in more than one box, which means that either there is
more than one pathway for degradation or that some of the carbon atoms of the skeleton emerge
as one product and others as another product.

Amino acids that are degraded to citric acid cycle intermediates can serve as glucose
precursors and are called glucogenic amino acids. While amino acids that are degraded to
acetyl CoA or acetoacetyl CoA can contribute to the formation of fatty acids or ketone bodies
and are called ketogenic amino acids. Amino acids that are degraded to pyruvate can be either
glucogenic or ketogenic, since pyruvate can be metabolized to either oxaloacetate (glucogenic)
or acetyl CoA (ketogenic). Only two amino acids are purely ketogenic: leucine and lysine. Nine
amino acids are both glucogenic and ketogenic: those degraded to pyruvate, as well as
tyrosine, phenylalanine, and isoleucine (which have two degradation products). The remaining
nine amino acids are purely glucogenic.

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The existence of glucogenicity and ketogenicity for amino acids points out that ATP production is
not the only fate for amino acid degradation products. They can also be converted to glucose,
ketone bodies, or fatty acids (via acetyl CoA).

CONCLUSION:
INTERRELATIONSHIPS AMONG METABOLIC PATHWAYS

We have considered thus far the metabolic pathways of carbohydrates, lipids, and proteins. It is
important to note that these pathways are not independent of each other but, rather, are integrally
linked. The numerous connections among pathways mean that a change in one pathway can
affect many other pathways. The figure below shows this connection.

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A good illustration of the interrelationships among pathways emerges from comparing the
processes of eating (feasting), not eating for a short period (fasting), and not eating for a
prolonged period (starvation). The figure shows how the body responds to each of these
situations.

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