NNAMDI AZIKIWE UNIVERSITY, AWKA

Glycolytic pathway, Tricarboxylic Acid Cycle (TCA cycle), Oxidative phosphorylation (electron

transport chain), Lipid metabolism, Amino Acid metabolism, Nucleic Acid metabolism.

SUBMITTED BY

MEZUE JACHIKE IFECHUKWU

2015874019

COURSE CODE: FAM 523

COURSE TITLE: AQUATIC MICROBIOLOGY

LECTURER: MR EVULOBI

JUNE, 2020
 ACKNOWLEDGEMENTS

I give thanks to the Almighty for seeing me through the period of learning, despite the ups and

downs, May his name be praised.

TABLE OF CONTENTS
CHAPTER ONE

1.0 GLYCOLYTIC PATHWAY

CHAPTER TWO

2.0 TRICARBOXYLIC ACIDIC CYCLE (KREB CYCLE)

CHAPTER THREE

3.0 OXIDATIVE PHOSPHORITION (Electron transport chain)

CHAPTER FOUR

4.0 LIPID METABOLISM

CHAPTER FIVE

5.0 AMINO ACID METABOLISM

CHAPTER SIX

6.0 NUCLEIC ACID METABOLISM

CHAPTER ONE
1.0 GLYCOLYSIS/ GLYCOLYTIC PATHWAY

Glycolysis is the process in which one glucose molecule is broken down to form two molecules
of pyruvic acid (also called pyruvate). The glycolysis process is a multi-step metabolic pathway
that occurs in the cytoplasm of animal cells, plant cells, and the cells of microorganisms. At least
six enzymes operate in the metabolic pathway.

Glycolysis is the metabolic process that serves as the foundation for both aerobic and anaerobic
cellular respiration. In glycolysis, glucose is converted into pyruvate. Glucose is a six-
memebered ring molecule found in the blood and is usually a result of the breakdown of
carbohydrates into sugars. It enters cells through specific transporter proteins that move it from
outside the cell into the cell’s cytosol. All of the glycolytic enzymes are found in the cytosol.

The overall reaction of glycolysis which occurs in the cytoplasm is represented simply as:

C6H12O6 + 2 NAD+ + 2 ADP + 2 P —–> 2 pyruvic acid, (CH 3(C=O)COOH + 2 ATP + 2

NADH + 2 H+
PLATE 1: GLYCOLYTIC PATHWAY SKETCH

STEP 1: HEXOKINASE

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

The reaction occurs with the help of the enzyme hexokinase, an enzyme that catalyzes the
phosphorylation of many six-membered glucose-like ring structures. Atomic magnesium (Mg) is
also involved to help shield the negative charges from the phosphate groups on the ATP
molecule. The result of this phosphorylation is a molecule called glucose-6-phosphate (G6P),
thusly called because the 6′ carbon of the glucose acquires the phosphate group.
STEP 2: PHOSPHOGLUCOSE ISOMERASE

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

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

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

STEP 3: PHOSPHOFRUCTOKINASE

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

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

The enzyme that catalyzes this reaction is phosphofructokinase (PFK). As in step 1, a

magnesium atom is involved to help shield negative charges.

STEP 4: ALDOLASE

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

STEP 5: TRIOSEPHOSPHATE ISOMERASE

GAP is the only molecule that continues in the glycolytic pathway. As a result, all of the DHAP
molecules produced are further acted on by the enzyme Triosephosphate isomerase (TIM), which
reorganizes the DHAP into GAP so it can continue in glycolysis. At this point in the glycolytic
pathway, we have two 3-carbon molecules, but have not yet fully converted glucose into
pyruvate.
STEP 6: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

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

The enzyme GAPDH contains appropriate structures and holds the molecule in a conformation
such that it allows the NAD molecule to pull a hydrogen off the GAP, converting the NAD to
NADH. The phosphate group then attacks the GAP molecule and releases it from the enzyme to
yield 1,3 bisphoglycerate, NADH, and a hydrogen atom.

STEP 7: PHOSPHOGLYCERATE KINASE

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

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

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

STEP 8: PHOSPHOGLYCERATE MUTASE

This step involves a simple rearrangement of the position of the phosphate group on the 3
phosphoglycerate molecule, making it 2 phosphoglycerate. The molecule responsible for
catalyzing this reaction is called phosphoglycerate mutase (PGM). A mutase is an enzyme that
catalyzes the transfer of a functional group from one position on a molecule to another.
The reaction mechanism proceeds by first adding an additional phosphate group to the 2′ position
of the 3 phosphoglycerate. The enzyme then removes the phosphate from the 3′ position leaving
just the 2′ phosphate, and thus yielding 2 phsophoglycerate. In this way, the enzyme is also
restored to its original, phosphorylated state.

STEP 9: ENOLASE

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

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

STEP 10: PYRUVATE KINASE

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

Immediately upon finishing glycolysis, the cell must continue respiration in either an aerobic or
anaerobic direction; this choice is made based on the circumstances of the particular cell. A cell
that can perform aerobic respiration and which finds itself in the presence of oxygen will
continue on to the aerobic citric acid cycle in the mitochondria. If a cell able to perform aerobic
respiration is in a situation where there is no oxygen (such as muscles under extreme exertion), it
will move into a type of anaerobic respiration called homolactic fermentation. Some cells such as
yeast are unable to carry out aerobic respiration and will automatically move into a type of
anaerobic respiration called alcoholic fermentation.
 CHAPTER TWO

2.0 TRICARBOXYLIC ACIDIC CYCLE (KREB CYCLE)

PLATE 2: TRICARBOXYLIC ACID CYCLE SKETCH

Tricarboxylic acid cycle, (TCA cycle), also called Krebs cycle and citric acid cycle, the

second stage of cellular respiration, the three-stage process by which living cells break down
organic fuel molecules in the presence of oxygen to harvest the energy they need to grow and
divide. This metabolic process occurs in most plants, animals, fungi, and many bacteria. In all
organisms except bacteria the TCA cycle is carried out in the matrix of intracellular structures
called mitochondria.
The TCA cycle plays a central role in the breakdown, or catabolism, of organic fuel molecules—
i.e., glucose and some other sugars, fatty acids, and some amino acids. Before these rather large
molecules can enter the TCA cycle they must be degraded into a two-carbon compound called
acetyl coenzyme A (acetyl CoA). Once fed into the TCA cycle, acetyl CoA is converted
into carbon dioxide and energy.

Functions of the Citric Acid Cycle

1. The final common oxidative pathway that oxidizesacetyl-CoA to CO2.

2. The source of reduced coenzymes that provide the substrate for the respiratory chain.

3. The link between catabolic and anabolic pathways (amphibolic role).

4. Provides precursors for synthesis of amino acids and nucleotides.

5. Components of the cycle have direct or indirect controlling effects on key enzymes of other
pathways.

Reactions of the Cycle

Preparatory Steps

Acetyl-CoA enters the cycle, and is completely oxidized. During this process, energy is trapped.
The sources of acetyl-CoA are shown in Figure 20.1. Pyruvate derived from glycolysis is
oxidatively decarboxylated to acetyl-CoA by the pyruvate dehydrogenase.

This is the link between the TCA cycle and glycolysis. The acetyl-CoA is also derived from
beta-oxidation of fatty acids. All the enzymes of citric acid cycle are located inside the
mitochondria.
FIRST STEP: FORMATION OF CITRIC ACID

The 4 carbon, oxaloacetate condenses with 2 carbons, acetyl-CoA to form 6 carbon compound,
the citrate (tricarboxylic acid). The enzyme is citrate synthase. The hydrolysis of the thioester
bond in acetyl-CoA drives the reaction forward. This is an irreversible step. However, body can
reverse this step by another enzyme, ATP-citrate lyase.

SECOND STEP: FORMATION OF ISOCITRATE

Citrate is isomerized to isocitrate by aconitase. This reaction takes place in two steps, withcis-
aconitate as the intermediary.

THIRD STEP: FORMATION OF ALPHA KETOGLUTARATE

This reaction is catalyzed by the enzyme, isocitrate dehydrogenase. First isocitrate is

dehydrogenated to form oxalosuccinate. It undergoes spontaneous decarboxylation to form alpha
ketoglutarate.

The NADH generated in this step is later oxidizedin electron transport chain (ETC) to generate
ATPs. Isocitrate (6 carbons) undergoes oxidative decarboxylation to form alpha ketoglutarate (5
carbons). In this reaction, one molecule of CO2 is liberated.

FOURTH STEP: FORMATION OF SUCCINYL-COA

Next, alpha ketoglutarate is oxidatively decarboxylated to form succinyl-CoA by the enzyme

alpha ketoglutarate dehydrogenase. The NADH thus generated enters into ETC to generate
ATPs. Another molecule of CO2 is removed in this step.

This is the only irreversible step in the whole reaction cycle. The enzyme alpha ketoglutarate
dehydrogenase is a multienzyme complex having 3 enzyme proteins and 5 coenzymes.
FIFTH STEP: GENERATION OF SUCCINATE

The next reaction involves a substrate level phosphorylation whereby a high energy phosphate is
generated from the energy trapped in the thioester bond of succinyl-CoA. The enzyme is
succinate thiokinase. A molecule of GDP is phosphorylated to GTP and succinate is formed. The
GTP can be converted to ATP by reacting with an ADP molecule:

GTP + ADP → GDP + ATP

SIXTH STEP: FORMATION OF FUMARATE

Succinate is dehydrogenated to fumarate, an unsaturated dicarboxylic acid, by succinate

dehydrogenase. The hydrogen atoms are accepted by FAD. The FADH2 then enters into ETC to
generate ATPs. The succinate dehydrogenase is competitively inhibited by malonate.

SEVENTH STEP: FORMATION OF MALATE

The formation of malate from fumarate is catalyzed by fumarase. The reaction involves the
addition of a water molecule.

EIGHTH STEP: REGENERATION OF OXALOACETATE

Finally malate is oxidized to oxaloacetate by malate dehydrogenase (step 8, Fig. 20.2). The
coenzyme is NAD+. The NADH is generated in this step, which enters the electron transport
chain, when ATPs are produced. The oxaloacetate can further condense with another acetyl-CoA
molecule and the cycle continues.
 CHAPTER THREE

3.0 OXIDATIVE PHOSPHORITION (ELECTRON TRANSPORT CHAIN)

PLATE 3: OXIDATIVE PHOSPHORELATION SKETCH

Oxidative phosphorylation is the process where energy is harnessed through a series of protein
complexes embedded in the inner-membrane of mitochondria (called the electron transport chain
and ATP synthase) to create ATP. Oxidative phosphorylation can be broken down into two parts:

1) Oxidation of NADH and FADH and

2) Phosphorylation.

STEP 1

Oxidative phosphorylation starts with the arrival of 3 NADH and 1 FADH transfers its high
energy molecules to protein complex 1,  while FADH transfers its high energy molecules to
protein complex 2. Shuttling high energy molecules causes a loss of electrons from NADH and
FADH, called oxidation (other molecules are also capable of being oxidized).

The opposite of oxidation is reduction, where a molecule gains electrons (which is seen in the

citric acid cycle). Here’s an easy way to remember which process gains or loses electrons:

STEP 2

The process of NADH oxidation leads to the pumping of protons (single positively-charged
hydrogen atoms denoted as H+ through protein complex 1 from the matrix to the inter membrane
space. The electrons that were received by protein complex 1 are given to another membrane-
bound electron carrier called ubiquinone or Q.

This process of transferring electrons drives the pumping of protons, which is seen as
unfavorable. Electron transfer driving proton pumping is repeated in complexes 3 and 4 (which
we will discuss in steps 2 - 5). As this action is repeated, protons will accumulate in the inter
membrane space. This accumulation of protons is how the cell temporarily stores transformed
energy.

STEP 3

The rest of the steps are now the same for the high energy molecules from NADH and FADH in
earlier steps. Inside the nonpolar region of the phospholipid bilayer, UQH (which is also a
nonpolar compound) transports the electrons to protein complex 3. UQH also carries protons.
When UQH delivers electrons to protein complex 3, it also donates its protons to be pumped.

STEP 4

The electrons that arrived at protein complex 3 are picked up by cytochrome C (or “cyt C”), the
last electron carrier. This action also causes protons to be pumped into the inter membrane space.

STEP 5

Cytochrome C carries the electrons to the final protein complex, protein complex 4. Once again,
energy released via electron shuttling allows for another proton to be pumped into the inter
membrane space. The electrons are then drawn to oxygen, which is the final electron acceptor.
It’s important to note that oxygen must be present for oxidative phosphorylation to occur. Water
is formed as oxygen receives the electrons from protein complex 4, and combines with protons
on the inside of the cell.

STEP 6

As a result of part 1 (Oxidation of NADH and FADH), an electrochemical gradient is created,

meaning there is a difference in electrical charge between the two sides of the inner
mitochondrial membrane. The outside, or exterior, of the mitochondrial membrane is positive
because of the accumulation of the protons (H+), and the inside is negative due to the loss of the
protons. A chemical concentration gradient has also developed on either side of the membrane.
The electrochemical gradient is how the cell transfers the stored energy from the reduced NADH
and FADH.

STEP 7

When there is a high concentration of protons on the outside of the mitochondrial membrane,
protons are pushed through ATP synthase. This movement of protons causes ATP synthase to
spin, and bind ADP and Pi, producing ATP. Finally, ATP is made.

CHAPTER FOUR

4.0 LIPID METABOLISM

PLATE 4: LIPI METABOLISM SKETCH

Lipid metabolism is the synthesis and degradation of lipids in cells, involving the breakdown or
storage of fats for energy and the synthesis of structural and functional lipids, such as those
involved in the construction of cell membranes.

Lipids are absorbed from the intestine and undergo digestion and metabolism before they can be
utilized by the body. Most of the dietary lipids are fats and complex molecules that the body
needs to break down in order to utilize and derive energy from.

The major aspects of lipid metabolism are involved with Fatty Acid Oxidation to produce
energy or the synthesis of lipids which is called Lipogenesis. Lipid metabolism is closely
connected to the metabolism of carbohydrates which may be converted to fats.

Metabolism of Lipids

Metabolism / Digestion of fats comprises of these major stages:-

1. Absorption

2. Emulsification of Fats

3. Digestion of Fats

4. Fat Metabolism

5. Degradation

ABSORPTION OF LIPIDS

Short-chain fatty acids (up to 12 carbons) are absorbed directly.

Triglycerides and dietary fats are insoluble in water and thus their absorption is difficult. To
achieve this, the dietary fat is broken down into small particles that increases the exposed area
for rapid attack by digestive enzymes.
EMULSIFICATION OF FATS

Dietary fats undergo emulsification that leads to liberation of fatty acids. This is brought about
by simple hydrolysis of the ester bonds in triglycerides.

Fats are broken down into small particles by detergent action and mechanical mixing. The
detergent action is performed by digestive juices, but especially by partially digested fats (fatty
acid soaps and monacylglycerols) and by bile salts.

The bile salts such as cholic acid contain a side that is hydrophobic (repellent to water) and
another water loving or hydrophhillic side. This allows them to dissolve at an oil-water interface,
with the hydrophobic surface in contact with the lipid to be absorbed and the hydrophilic surface
in the watery medium. This is called the detergent action and this emulsifies fats and yields
mixed micelles.

Mixed Micelles serve as transport vehicles for less water soluble lipids from food and also for
cholesterol, fat-soluble vitamins A, D, E, and K.

DIGESTION OF FATS

After emulsification the fats are hydrolyzed or broken down by enzymes secreted by the
pancreas. The most important enzyme involved is pancreatic lipase. Pancreatic lipase breaks
down primary ester linkages, the 1 or the 3 ester bonds. This converts triglycerides to 2-
monoglycerides (2-monoacylglycerols). Less than 10% of triglycerides remain unhydrolyzed in
the intestine.

FAT METABOLISM

Short chain fatty acids enter the circulation directly but most of the fatty acids are reesterified
with glycerol in the intestines to form triglycerides that enter into the blood as lipoprotein
particles called chylomicrons.
Lipoprotein lipase acts on these chylomicrons to form fatty acids. These may be stored as fat in
adipose tissue, used for energy in any tissue with mitochondria using oxygen and reesterified to
triglycerides in the liver and exported as lipoproteins called VLDL (very low density
lipoproteins).

VLDL has a similar outcome as chylomicrons and eventually is converted to LDL (low density
lipoproteins). Insulin simulates lipoprotein lipase.
During starvation for long periods of time the fatty acids can also be converted to ketone bodies
in the liver. These ketone bodies can be used as an energy source by most cells that have
mitochondria.

DEGRADATION

Fatty acids are broken down by Beta oxidation. This occurs in the mitochondria and/or in
peroxisomes to generate acetyl-CoA. The process is the reverse of fatty acid synthesis: two-
carbon fragments are removed from the carboxyl end of the acid. This occurs after
dehydrogenation, hydration, and oxidation to form a beta-keto acid.

The acetyl-CoA then converts to ATP, CO2, and H2O using the citric acid cycle and releases
energy of 106 ATP. Unsaturated fatty acids require additional enzymatic steps for degradation.
 CHAPTER FIVE

5.0 AMINO ACID METABOLISM

PLATE 5: AMINO ACID METABOLISM SKETCH

Amino acid synthesis is the set of biochemical processes (metabolic pathways) by which
the amino acids are produced. The substrates for these processes are various compounds in
the organism's diet or growth media. Not all organisms are able to synthesize all amino acids

Of the basic set of twenty amino acids (not counting selenocysteine), humans cannot synthesize
eight.
In addition, the amino acids arginine, cysteine, glycine, glutamine, histidine, proline, serine,
and tyrosine are considered conditionally essential, meaning they are not normally required in
the diet but must be supplied exogenously to specific populations that do not synthesize it in
adequate amounts. For example, enough arginine is synthesized by the urea cycle to meet the
needs of an adult but perhaps not those of a growing child. Amino acids that must be obtained
from the diet are called essential amino acids. Nonessential amino acids are produced in the
body. The pathways for the synthesis of nonessential amino acids are quite simple. Glutamate
dehydrogenase catalyzes the reductive amination of α-ketoglutarate to glutamate.
A transamination reaction takes place in the synthesis of most amino acids. At this step, the
chirality of the amino acid is established. Alanine and aspartate are synthesized by the
transamination of pyruvate and oxaloacetate, respectively. Glutamine is synthesized from NH4+
and glutamate, and asparagine is synthesized similarly. Proline and arginine are derived from
glutamate. Serine, formed from 3-phosphoglycerate, is the precursor
of glycine and cysteine. Tyrosine is synthesized by the hydroxylation of phenylalanine, an
essential amino acid. The pathways for the biosynthesis of essential amino acids are much more
complex than those for the nonessential ones.

ESSENTIAL AND NON-ESSENTIAL AMINO ACID

Human body is able to synthesize only certain amino acids (non-essential, dispensable).  The
other amino acids (essential, indispensable) must receive in food. Here’s an overview:

Essential amino acids

1) Branched: Val, Leu, Ile

2) Aromatic: Phe, Trp

3) Basic: Lys

4) Sulphur-containing: Met

5) With hydroxyl group: Thr

Conditionally essential amino acids

Arg, His

Non-essential amino acids

Gly, Ala, Ser, Pro, Cys, Tyr, Asn, Gln, Asp, Glu

Important reactions in amino acid metabolism

1) Decarboxylation means a removal of the carboxyl group – biogenic amines are formed

2) Transamination means an exchange of amino group with 2-oxoacid – 2-oxoacids are formed

3) Oxidative deamination means an oxidative removal of amino group – 2-oxoacids are formed

4) Peptide bond formation – peptide and protein generation

Defects in amino acid metabolism

In humans, there are many different genetic defects in amino acid metabolism. Some
their intermediates accumulate in the body causing defective development of the nervous
system that often results in mental retardation.

Removal of amino group

Removal of amino group is a crucial step in the amino acid catabolism. The nitrogen of the
amino groups (amino nitrogen) cannot be used for energy production and must be removed from
our body. The first way is an amino nitrogen conversion to a urea (about 95%), followed by urea
excretion from the body via the urine. The second way is amino nitrogen releasing from
glutamine in the form NH3/NH4+ in the tubular cells of the kidney (about 5%).

Transamination

Transaminations are freely reversible reactions catalyzed

by transaminases (aminotransferases). Amino group of α-amino acid is exchanged with oxo
group of 2-oxoacid during transamination – from the amino acid, this produces 2-oxoacid, while
from the original 2-oxoacid, amino acid is formed.

The amino group is transferred by cofactor pyridoxal phosphate (PLP, derivative of  vitamin B6)

to oxoacid (Schiff´s base formation).

Most of the amino acids undergo transamination in their degradation. Enzymes aspartate

aminotransferase (AST) and alanine aminotransferase (ALT) are concrete examples of
transaminases that are normally detected as markers of potential damage to the liver
cells. Catalyzed reactions of AST and ALT are presented in the scheme.

The resulting 2-oxo acids (oxaloacetate and pyruvate) are involved in energy metabolism within
cells.

But there are exceptions (e.g. threonine) that is not degraded by transamination.

Glutamate / glutamine conversion

Conversion of carboxy group in glutamate (in the side chain) to amide group in glutamine is
catalyzed by cytosolic enzyme glutamine synthetase. ATP and NH4+ are also needed. This
reaction is used in the cells of the CNS as the main detoxification mechanism removing toxic
NH3 from brain tissue.  Emerging glutamine is the most important transport form of amino
nitrogen (ammonia) in the blood – provides transport from extrahepatic tissues by the blood to
the liver and kidneys. Glutamine has the highest plasma concentration of the amino acids – 0.6
mmol/l (alanine – 0.3 mmol/l). Two amino groups / ammonia are “stored” in glutamine
molecule. Glutamine is able to bring the ammonia to different biosynthetic processes – for
example in synthesis of purine bases.

Mitochondrial enzyme glutaminase catalyzes the release of NH3 from glutamine (hydrolytic

deamination, frequently occurs in hepatocytes and kidney tubule cells). The ammonia produced
in the liver mitochondria enters the urea cycle. In the kidneys, ammonia is excreted in the urine,
where it serves as a buffer.

OXIDATIVE DEAMINATION

During oxidative deamination amino group is converted to keto group with simultaneous release
of NH3. Glutamate is the only one amino acid that is deaminated with sufficient speed in the
human body. Glutamate dehydrogenase catalyzing the oxidative deamination of glutamate  is
found in the mitochondrial matrix (mainly liver cells). This process demonstrates the following
reaction:

Glutamate + NAD+ → α-ketoglutarate + NH4+ + NADH + H+

The resulting NH4+ enters the urea cycle and α-ketoglutarate may be used in the transamination
or Krebs cycle. The mentioned reaction is fully reversible – glutamate can be synthesized from
α-KG and NH4+ .

We can conclude that most of the amino acid undergoes transamination in its degradation and
that the majority of amino nitrogen from amino acids is directly or indirectly concentrated in the
molecule of glutamate / glutamine. Amino nitrogen is subsequently released in glutaminase and
glutamate dehydrogenase reaction.

UREA (ORNITHINE) CYCLE

AMMONIA TOXICITY

Ammonia is a polar substance freely passing through physical barriers, as well as the blood-brain

barrier. When its concentration increases in the body, balance of many important reactions is
altered. Consider the following examples:

Glutamate + NAD+ → α-ketoglutarate + NH4+

Glutamate  + NH4+ + ATP → glutamine + ADP + Pi

When an excess of ammonia, the glutamine concentration is gradually increasing. But glutamine

formation also consumes α-ketoglutarate of the Krebs cycle – speed of this pathway is gradually
decreasing and thus the production of energy in cells. The plasma ammonia concentration should
not exceed 35 μmol / L.  In the human body, most of the toxic ammonia is converted to urea by
reactions of urea cycle.

Reactions in urea cycle

Urea, a non-toxic compound, is transported via the bloodstream to the kidneys where it is
excreted with the urine. Urea cycle is located in the matrix of mitochondria and cytosol of liver
cells. This pathway is an energy-consuming process in which the three substrates enter
– ammonia, carbon dioxide (bicarbonate) and aspartate (its amino group). Mitochondrial
carbamoyl phosphate synthetase I is the regulatory enzyme. Ornithine cycle communicates with
the Krebs cycle via oxaloacetate and fumarate.

Urea formation involves five reactions:

1) Carbamoyl phosphate formation is catalyzed by mitochondrial carbamoyl phosphate

synthetase I:

NH4+ + HCO3– + ATP → carbamoyl phosphate + 2 ADP + Pi

2) Citrulline formation is catalyzed by ornithine transcarbamoylase:

Ornithine + carbamoyl phosphate → citrulline + Pi

Citrulline is passed into the cytosol.

3) Argininosuccinate formation is catalyzed by argininosuccinate synthetase:

Citrulline + Asp + ATP → argininosuccinate + AMP + PPi

4) Argininosuccinate break down is catalyzed by argininosuccinate lyase:

Argininosuccinate → arginine + fumarate

5) Hydrolysis of arginine is catalyzed by arginase:

Arginine + H2O → ornithine + urea

Ornithine returns into the mitochondrial matrix.

The urea cycle is closely linked to the Krebs cycle – from emerging fumarate becomes aspartate.
How does this relationship work? The fumarate is first hydrated to malate which is converted to
oxaloacetate by oxidation. Enzyme aspartate aminotransferase catalyzes transamination between
glutamate and oxaloacetate, resulting aspartate enters ornithine cycle. Glutamate is produced by
transamination of degraded amino acids which transmit their amino groups on the α-
ketoglutarate molecule.

REGULATION OF ORNITHINE CYCLE

Carbamoyl phosphate synthetase I, the main regulatory enzyme of ornithine cycle, is activated
by N-acetylglutamate. Enzyme N-acetylglutamate synthetase catalyzes the reaction between
AcCoA and glutamate which produces N-acetylglutamate. The amino acid arginine increases the
enzyme activity. Transcription of urea cycle enzymes is increased in high-protein diet or
by increasing protein catabolism (e.g. starvation), therefore in the increased supply of amino
acids.  The urea cycle belongs among proton-producing reactions, its activity is reduced at lower
pH – acidosis.

GLUCOSE-ALANINE CYCLE
Alanine participates in the transmission of blood ammonia and also serves through pyruvate as a
significant source of carbons in gluconeogenesis – see Subchapter 2/9. Glucose-alanine cycle is
pathway extending between the liver and muscle cells.  Pyruvate produced in muscle cells
undergoes the transamination to give alanine. It is released into the blood and transferred to the
liver where it is converted back to pyruvate by transamination that can be involved in the process
of gluconeogenesis.  The resulting glucose is released into the blood and enters muscle cells and
the cycle continues. Transferred amino group (ammonia) is directed to the urea cycle.

DEGRADATION OF AMINO ACID CARBON SKELETONS

Proteins in the human body contain 20 (21 if we include selenocysteine) proteinogenic amino
acids. Twenty (twenty-one) different multienzyme sequences exist for catabolism of amino acid
carbon skeletons. In this text we restrict ourselves only to the basic general mechanisms of amino
acid carbon skeleton degradation and a few selected examples.

 Catabolism of amino acid carbon skeletons results in the formation of seven

products: pyruvate, acetyl-CoA, acetoacetyl-CoA, α-ketoglutarate, suc-CoA, fumarate and oxalo
acetate. They have a different fate in the energy metabolism. The strategy of the cell is to convert
amino acid carbon skeletons to compounds useful in gluconeogenesis or a molecule of lipids
(fatty acids and ketone bodies). Amino acids are divided into glucogenic and ketogenic amino
acids according to the fate of their degradation products. Amino acids leucine and lysine (starting
with the letter L) belong among ketogenic amino acids which lead to the formation of acetyl-
CoA and acetoacetyl-CoA. Glucogenic amino acids include those that lead to the formation of
the remaining five products – pyruvate, α-ketoglutarate, suc-CoA, fumarate or oxaloacetate –
serine, threonine, cysteine, methionine, aspartate, glutamate, asparagine, glutamine, glycine,
alanine, valine, proline, histidine and arginine. But some amino acids have two degradation
products – one of them being glucogenic and second one ketogenic. These amino acids are
called keto- and glucogenic amino acids – they include isoleucine, phenylalanine, tyrosine and
tryptophan.
The following overview shows degradation products of particular amino acids:

1) Acetyl-CoA and acetoacetyl-CoA – Lys and Leu are purely ketogenic amino acids, some
other amino acids (Phe, Tyr, Trp, Ile) provide glucogenic and ketogenic degradation products

2) α-ketoglutarate – five-carbon amino acids – Glu, Gln, Pro, Arg a His

3) Suc-CoA – nonpolar amino acids – Met, Ile a Val

4) Fumarate – Phe, Tyr

5) Oxaloacetate – four-carbon amino acids – Asp a Asn

6) Pyruvate – Cys, Ala, Ser, Gly, Thr, Trp

Degradation of branched amino acids – Val, Leu and Ile

These amino acids are not degraded in the liver cells, but mainly in extrahepatic tissues –
especially high activity in muscle cells. They contain specific transaminase producing the
appropriate α-keto acids – the so-called keto analogs of branched amino acids. This transaminase
is not present in liver cells. Keto analogs are converted to acyl-CoA derivatives
by a dehydrogenation complex which catalyzes the oxidative
decarboxylation and dehydrogenation.

The genetic defect of dehydrogenation complex causes a maple syrup urine disease. This
relatively rare disease can lead to the accumulation of the appropriate α-keto acids in tissues and
body fluids (urine smells like caramel). The defect causes abnormal brain development, mental
retardation and can result in death of the individual.

Formation of non-essential amino acids in the human body

The human body is not able to synthesize essential amino acids – Phe, Trp, Val, Leu, Ile, Met,
Thr and Lys. Two amino acids are essential in the growth of the organism (the period of their
increased consumption), the rate of their synthesis is not sufficient to cover the claims of the
body – the so-called conditionally essential amino acids – Arg, His. Other amino acids belong to
non-essential amino acids. Here is the overview of amino acid precursors:

1) Oxaloacetate → Asp, Asn

2) α-ketoglutarate → Glu, Gln, Pro, (Arg and His)

3) Pyruvate → Ala

4) 3-phosphoglycerate → Ser, Cys and Gly

5) Phe → Tyr

PHENYLKETONURIA (PKU)

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder (incidence 8-10 cases / 100

000 individuals) conditioned by the absence or reduced phenylalanine hydroxylase
activity. Physiologically this enzyme catalyzes hydroxylation of Phe to Tyr. The defective
enzyme leads to the phenylalanine accumulation and an alternative degradation of
Phe – phenylpyruvate (transamination), phenyllactate, phenylacetate and phenylethylamine are
formed. Such substances accumulate in tissues and body fluids and produce typical odor of urine
(mousy smell). Some of them cause severe brain damage. Phenylketonuria was the first
discovered human genetic defect in amino acid metabolism and is currently one of the diseases
for which a screening is carried out in all newborns. If it is detected even at this age, we can
prevent brain damage with a special diet low in Phe.
 CHAPTER SIX

6.0 NUCLEIC ACID METABOLISM

PLATE 6: NUCLEIC ACID METABOLISM PATHWAY SKETCH

Nucleic acid metabolism is the process by which nucleic acids (DNA and RNA) are synthesized
and degraded. Nucleic acids are polymers of nucleotides. Nucleotide synthesis is an anabolic
mechanism generally involving the chemical reaction of phosphate, pentose sugar, and a
nitrogenous base. Destruction of nucleic acid is a catabolic reaction. Additionally, parts of the
nucleotides or nucleobases can be salvaged to recreate new nucleotides. Both synthesis and
degradation reactions require enzymes to facilitate the event. Defects or deficiencies in these
enzymes can lead to a variety of diseases.

Nucleotides are organic molecules that serve as the monomer units for forming the nucleic acid
polymers deoxyribonucleic acid(DNA) and ribonucleic acid (RNA), both of which are essential
biomolecules within all life-forms on Earth. Nucleotides are the building blocks of nucleic acids;
they are composed of three subunit molecules: a nitrogenous base (also known as nucleobase), a
five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. A nucleoside is a
nitrogenous base and a 5-carbon sugar. Thus a nucleoside plus a phosphate group yields a
nucleotide.

PURINES & PYRIMIDINES ARE DIETARILY NONESSENTIAL: Human tissues can

synthesize purines and pyrimidines from intermediates. Ingested nucleic acids and nucleotides,
which therefore are dietary nonessential, are degraded in the intestinal tract to mononucleotides,
which may be absorbed or converted to purine and pyrimidine bases. The purine bases are then
oxidized to uric acid, which may be absorbed and excreted in the urine.

BIOSYNTHESIS OF PURINE NUCLEOTIDES Purine and pyrimidine nucleotides are

synthesized in vivo at rates consistent with physiologic need. Intracellular mechanisms sense and
regulate the pool sizes of nucleotide triphosphates, Three processes contribute to purine
nucleotide biosynthesis. These are, in order of decreasing importance:

(1) Synthesis from intermediates (synthesis de novo),

(2) Phosphoribosylation of purines, and

(3) Phosphorylation of purine nucleosides.

“SALVAGE REACTIONS” CONVERT PURINES & THEIR NUCLEOSIDES TO

MONONUCLEOTIDES.

Conversion of purines, their ribonucleosides, and their deoxyribonucleosides to mononucleotides

involves so called “salvage reactions” that require far less energy than de novo synthesis. The
more important mechanism involves: 1- phosphoribosylation by PRPP(5-phosphoribosyl -1-
pyrophosphate ) of a free purine (Pu) to form a purine 5′-mononucleotide. 2- Synthesis of 5-
phosphoribosylamine: Synthesis of 5'-phosphoribosylamine from PRPP and glutamine.

DEGRADATION OF PURINE NUCLEOTIDES Degradation of dietary nucleic acids occurs

in the small intestine, where a family of pancreatic enzymes hydrolyzes the nucleotides to
nucleosides and free bases. Inside cells, purine nucleotides are sequentially degraded by specific
enzymes, with uric acid as the end product of this pathway. [Note: Mammals other than primates
oxidize uric acid further to allantoin, which, in some animals other than mammals, may be
further degraded to urea or ammonia.].

A. DEGRADATION OF DIETARY NUCLEIC ACIDS IN THE SMALL INTESTINE

Ribonucleases and deoxyribonucleases, secreted by the pancreas, hydrolyze RNA and DNA
primarily to oligonucleotides. Oligonucleotides are further hydrolyzed by pancreatic
phosphodiesterases, producing a mixture of 3'- and 5'-mononucleotides. A family of
nucleotidases removes the phosphate groups hydrolytically, releasing nucleosides that may be
absorbed by the intestinal mucosal cells, or be further degraded to free bases before uptake.
[Note: Dietary purines and pyrimidines are not used to a large extent for the synthesis of tissue
nucleic acids. Instead, the dietary purines are generally converted to uric acid by intestinal
mucosal cells. Most of the uric acid enters the blood, and is eventually excreted in the urine. For
this reason, individuals with a tendency toward gout should be careful about consuming foods
such as organ meats, anchovies, sardines, or dried beans, which contain high amounts of nucleic
acids.

B. FORMATION OF URIC ACID

A summary of the steps in the production of uric acid and genetic diseases associated with
deficiencies of specific degradative enzymes.

1. An amino group is removed from AMP to produce IMP or from adenosine to produce
inosine (hypoxanthine ribose) by AMP or adenosine deaminase.
2. IMP and GMP are converted into their nucleoside forms – inosine and guanosine –by the
action of 5’nucieotidase.
3. Purine nucleoside phosphorylase converts inosine and guanosine into their respective
puriene bases, hypoxanthine and guanine
4. Guanine is delaminated to form xanthine
 5. Hypoxanthine is oxidized by xanthine oxidase to xanthine, which is further oxidized by
xanthine oxidase to uric acid, the final product of human purine degradation. Uric acid is
excreted in the urine.

REFERENCES

Lehninger Principles of Biochemistry. 5th Edition. Chapter 15 : The Citric Acid Cycle.

Clackamas Community College. Citric Acid Cycle. Shmoop. Citric Acid Cycle.
Gary E. Kaiser. Community College of Baltimore County. The Citric (Krebs) Acid Cycle.
University of Michigan. Krebs Cycle.

Newcastle University – Teaching Server. School of Biomedical Sciences Wiki. Krebs cycle.

Spark Notes. The Citric Acid Cycle. John M. WyzAnt, Inc. The Krebs Cycle.

University of California, Davis. Chem Wiki. Kreb’s Cycle.

Boundless Biology. Citric Acid Cycle. Chemistry Learning. Krebs Cycle.

Khan Academy. Krebs / citric acid cycle.

Tutor Vista. Biology. Citric Acid Cycle. Study.com. The Citric Acid (Krebs) Cycle: Products
and Steps.

Wikipedia. Citric Acid Cycle.

Lehninger, Principles of Biochemistry, 5th edition, Chapter 18

Marks’ Medical Biochemistry, Section 7

Source: http://seqcore.brcf.med.umich.edu/mcb500/aametov.html