Mighty T

GLYCOLYSIS
(EMBDEN-MEYERHOF PATHWAY)

Glycolysis is the sequential degradation of a molecule of glucose(6-carbon compound) in a series of enzyme-

catalyzed reactions to yield two molecules of the three-carbon compound( pyruvate) .
It is derived from two Greek words, “glykys” meaning “sweet” and” lysis” meaning” split”.

Site of reaction: All the reaction steps take place in the cytoplasm.

The pathway is also known as EMBDEN-MEYERHOF PATHWAY due to the immense contribution of
Gustave Emden who studied the lactic acid formation from pyruvate and Otto Meyerhof who enunciated
most of the steps of the glycolytic pathway(Nobel prize,1922).
Glycolysis was the first metabolic pathway to be elucidated and is probably the best understood. During the
sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of
ATP and NADH.
Glycolysis is an almost universal central pathway of glucose catabolism, the pathway with the largest flux of
carbon in most cells.
Importance of glycolytic pathway

1. It is the sole source of metabolic energy in some mammalian tissues and cell types (erythrocytes, renal
medulla, brain, and sperm).
2. It is the only pathway that is taking place in all the cells of the body.
3. The glycolytic pathway provides carbon skeletons for the synthesis of non-essential amino acids as
well as glycerol part of fat.
4. Most of the glycolysis reactions are reversible, which are also used for glucose synthesis
(gluconeogenesis).
5. Anaerobic glycolysis forms the major source of energy for muscles during strenuous exercise.
6. It can be oxidized to yield ribose-5-phosphate for nucleic acid synthesis via the pentose phosphate
pathway

Phases of Glycolysis

Glycolysis has two phases, namely:

1. Preparatory phase-Energy investment(which constitute the first five steps(step 1-5) in glycolytic
pathway)
2. Payoff phase –Energy yield(which constitute the last five steps(step 6-10) in glycolytic pathway)

Chemical transformation in glycolysis

In the sequential reactions of glycolysis, three type of chemical transformation are particularly noteworthy:
(a) Degradation of carbon skeleton of glucose to yield pyruvate
(b) Phosphorylation of ADP to ATP by high energy phosphate compound form during glycolysis.
(c) Transfer of a hydride ion to NAD+ forming NADH.

Fates of Glucose
Glucose has three major fates;
1. It may be oxidized to a three carbon compound(pyruvate) via glycolysis to provide ATP and metabolic
intermediate
2. Oxidized via the pentose phosphate (phosphogluconate) pathway to yield ribose-5-phosphate for
nucleic acid synthesis.
3. It is stored as polysaccharides or sucrose

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Steps in glycolysis
• Preparatory phase
1. Phosphorylation of Glucose: In the first step of glycolysis, glucose is activated for subsequent
reactions at C-6 to yield glucose-6-phosphate, with ATP as the phosphoryl donor.

This irreversible reaction is catalyzed by hexokinase. Note that kinases are enzymes that catalyze the transfer
of the terminal phosphoryl group from ATP to an acceptor nucleophile. The type of hexokinase present in
hepatocytes(liver cells) is called hexokinase IV or glucokinase.

2. Conversion of Glucose-6-phosphate to fructose-6-phosphate:Phosphohexose isomerase

(phosphoglucose isomerase) catalyzes the reversible isomerization of glucose-6-phosphate (aldose)
to fructose-6-phosphate (a ketose)

3. Phosphorylation of fructose-6-phosphate to fructose 1,6 Bisphosphate: This is the second priming

reaction catalyzed by phosphofructokinase-1(PFK-1). This enzyme catalyzes the transfer of a
phosphoryl group from ATP to fructose-6-phosphate to fructose 1,6- bisphosphate. The reaction
catalyzed by this enzyme is the first committed step in the glycolytic pathway.

4. Cleavage of fructose 1,6- Bisphosphate: The enzyme fructose 1,6-bisphosphate aldolase, often
called simply aldolase, catalyzes a reversible aldol condensation. Fructose 1,6- bisphosphate is cleaved
to yield two different triose phosphates, glyceraldehyde 3-phosphate, an aldose, and
dihydroxyacetone phosphate, a ketose.

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5. Interconversion of the Triose phosphates: only one of the two triose phosphates formed by aldolase,
glyceraldehyde 3-phosphate, can be directly degraded in the subsequent steps of glycolysis. The other
product, dihydroxyacetone phosphate, is rapidly and reversibly converted to glyceraldehyde 3-
phosphate by the enzyme, triose phosphate isomerase.

• The payoff phase

6. Oxidation of glyceraldehyde 3-phosphate to 1,3-Bisphosphoglycerate: The first step in the payoff

phase is the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by
glyceraldehyde 3-phosphate dehydrogenase:

7. Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP: This reversible reaction catalyzed by

phosphoglycerate kinase involves the transfer of high-energy phosphoryl from the carboxyl group of
1,3-bisphosphoglycerate to ADP, forming 3-phosphoglycerate and ATP.

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 The formation of ATP by phosphoryl group transfer from a substrate such as 1,3-bisphosphoglycerate
is referred to a substrate level phosphorylation.

Mighty T

8. Conversion of 3-phosphoglycerate to 2-phosphoglycerate: The enzyme phosphoglycerate mutase

catalyzes a reversible shift (mutation)of the phosphoryl group between the C-2 and C-3 of glycerate;

9. Dehydration of 2-phosphoglycerate to Phosphoenol pyruvate: The enzyme enolase promotes the

reversible removal of a molecule of water from 2-phosphoglycerate to yield Phosphoenol
pyruvate(PEP).

10. Transfer of the phosphoryl group from Phosphoenol pyruvate to ADP: The last step in glycolysis
is also a substrate level phosphorylation which involves the transfer of the phosphoryl group from
Phosphoenolpyruvate to ADP is catalyzed by pyruvate kinase forming Pyruvate and ATP

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 Energy yield in glycolysis

• Energy yield(number of ATP generated) per molecule of glucose in the glycolytic pathway under
anaerobic conditions(oxygen deficiency)

Step Enzyme Source No. Of ATPs gained per

glucose molecule

1 Hexokinase —— –1

3 Phosphofructokinase —–– –1

7 1,3-bisphosphoglycerate ATP 1×2=2

kinase

10 Pyruvate kinase ATP 1×2=2

Total=4-2=2

• Energy yield Per molecule of glucose in the glycolytic pathway under aerobic conditions (oxygen
is available)

No. of ATPs gained per

Step Enzyme Source glucose molecule

1 Hexokinase ––– –1

3 Phosphofructokinase ––– –1

6 Glyceraldehyde-3- NADH 3×2=6

phosphate dehydrogenase

7 1,3-bisphosphoglycerate ATP 1×2=2

kinase

10 Pyruvate kinase ATP 1×2=2

Total=10–2 =8

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 • Energy yield per molecule of glucose when it completely oxidised through glycolysis plus Citric
acid cycle, under aerobic conditions
Pathway Step Enzyme Source No. of ATPs

Glycolysis 1 Hexokinase – –1

3 PFK-1 – –1

6 Glyceraldehyde-3- NADH 3×2=6

phosphate DH
7 1,3-BPG kinase ATP 1×2=2

10 Pyruvate kinase ATP 1×2=2

Pyruvate to acetyl Pyruvate DH NADH 3×2=6

CoA
TCA Cycle 3 Isocitrate DH NADH 3×2=6

4 alpha- NADH 3×2=6

ketoglutarate DH
5 Succinyl-CoA GTP 1×2=2
synthetase
6 Succinate DH FADH2 2×2=4

8 Malate DH NADH 3×2=6

Total=40-2=38

Note: NADH=3 or 2.5ATP and FADH2=2 or 1.5ATP

FATE OF PYRUVATE
Pyruvate under anaerobic condition(fermentation) is reduced to
I. Lactate
II. Ethanol.
• Under aerobic condition, it is oxidized to
III. Acetyl-CoA
1. Lactate Formation

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 When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of
pyruvate and NADH produced in glycolysis,NAD+ is regenerated from NADH by the reduction
of pyruvate to lactate. Tissues such as erythrocytes that lack mitochondria cannot oxidize
pyruvate to CO2( Krebs cycle) and thus produce lactate from glucose even under aerobic

conditions . This reaction is catalyzed by lactate dehydrogenase.

2. Ethanol formation: Yeast and other microorganisms ferment glucose to ethanol and CO2, rather than
to lactate. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate.
In the first step pyruvate is decarboxylated in a reaction catalyzed by pyruvate decarboxylase forming
acetaldehyde which is later reduced to ethanol by alcohol dehydrogenase

3. Acetyl-CoA formation: In the presence of oxygen, pyruvate is oxidized, with loss of its carboxyl
group as CO2 to yield the acetyl group of acetyl-coenzyme A(acetyl coA); the acetyl group is then
oxidized completely to CO2 by the citric acid cycle.

Relationship between Magnesium ion(Mg2+) and Adenosine triphosphate(ATP)

Magnesium is an essential element in biological systems, and it is present in every Organism. Adenosine
triphosphate(ATP), the main Source of energy in all cells must bind to a magnesium ion in order to be
biologically active. ATP is often called Mg-ATP(MgATP2-). Magnesium play an important role in the
stability of all polyphosphate in the cells, including those associated with the synthesis of DNA and RNA. It
play an important role in the glycolysis.
Magnesium ions associate with the negatively charged phosphate groups. This association facilitates the
nucleophilic attack of the phosphate group, leading to the transfer on to the hexose sugar.

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Regulation of Glycolytic pathway
The regulation of glycolysis is through the action of the enzymes catalyzing the irreversible steps;
• Glucokinase has a higher Km for glucose than hexokinase. Because of this low affinity, glucokinase can only
act when there is plenty supply of glucose. Thus, when the supply of glucose is limited, glucose-6-phosphate
will inhibit hexokinase.

• Phosphofructokinase(PFK-1) is the most important rate limiting enzyme for glycolysis. ATP and
Citrate are the most important allosteric inhibitors. AMP acts as an allosteric activator. Fructose-2,6-
bisphosphate increases the activity of PFK. F-2,6-BP is formed from fructose-6-phosphate by the
action of PFK-2.

• Pyruvate kinasecatalyses an irreversible step and is a regulatory enzyme of glycolysis. When energy
is plenty in cell, glycolysis is inhibited. Insulin increases its activity whereas glucagon inhibits.
Pyruvate kinase is inactive in the phosphorylated state.

• Hormonal regulation
Insulin favours glycolysis by activating the above three key glycolytic enzymes
Glucagon inhibit glycolysis and favours gluconeogenesis.

Past questions on glycolysis

• Discuss the fate of glucose in Embden-meyerhoff pathway.

• Explain how and why NAD+ is being regenerated in glycolysis
• Give the ten enzymatic reactions in Embden-meyerhoff pathway
• The irreversibility of pyruvate dehydrogenase reaction prelude the formation of pyruvate from acetyl-
CoA, discuss! Explain why glucose cannot be formed from acetyl-CoA.
• Name the inorganic substrate level phosphorylation in glycolysis.
• Name the coenzymes and prosthetic groups of pyruvate dehydrogenase.
• State the number of ATP generated in glycolysis
• Discuss briefly the fates of pyruvate.
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Mighty T

Gluconeogenesis
This is the process by which glucose molecules are synthesized from non-carbohydrate Precursors like lactate
and glucogenic amino acids. The Brain alone requires about 120g of glucose each day, and the supply of
glucose from its stored form(glycogen) is not always sufficient; between meals and during longer fasts, or
after vigorous exercise. Therefore organisms need a method for synthesizing glucose from non-carbohydrate
precursors accomplished through a pathway called gluconeogenesis. Glucose from the blood is the sole or
major fuel source in human Brain, nervous system, erythrocytes, testes, renal medulla, and embryonic
tissues.
Site of reaction: it occurs mainly in the mammalian liver, and to a lesser extent in the renal cortex. The
pathway is partly mitochondrial and partly cytoplasmic.
Gluconeogenesis and glycolysis are not identical pathways running in opposite directions, although they do
share several steps (seven of the ten enzymatic reactions of gluconeogenesis are the reverse of glycolysis).
Three reactions(1,3 and 10) of the glycolysis are essentially irreversible in vivo and cannot be used in
gluconeogenesis.
Importance of Gluconeogenesis
1. Replenish blood sugar
Gluconeogenesis helps in the replenish of blood sugar; an action only performed by liver because glucose 6-
phosphatase is present mainly in liver. So liver plays the major role in maintaining the blood glucose level.

2. During starvation
The stored glycogen is depleted within the first 12-18 hours of fasting; gluconeogenesis provides the
glucose needed for energy production. Gluconeogenesis is speeded up by substrate provided by protein
catabolism(glucogenic amino acids) and lactate.

3. Generation of glucose
In mammals(human brain, nervous system, erythrocytes, testes, renal medulla, and embryonic tissues
depend majorly on the glucose(energy) synthesized by gluconeogenesis.
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 Substrates for gluconeogenesis

i. Lactate
ii. Glucogenic amino acids
iii. Glycerol
iv. Propionyl CoA
Important: Mammals cannot use acetyl-CoA as a precursor of glucose, because the pyruvate dehydrogenase
reaction is irreversible and cells have no other pathway to convert acetyl-CoA to pyruvate, but microorganism
can do this conversion through gloxylate pathway.
Key glucogenic enzymes
• Pyruvate carboxylase
• Phosphoenol pyruvate carboxykinase
• fructose 1,6-bisphosphatase
• Glucose 6-phosphatase

Bypass reactions of gluconeogenesis

1. Conversion of pyruvate to Phosphoenol pyruvate: The first bypass reactions in gluconeogenesis is
the conversion of pyruvate to Phosphoenol pyruvate. This reaction is irreversible and has a large ,
negative standard free-energy change and thus cannot be reversed by the action of pyruvate kinase of
glycolysis.
Pyruvate is first transported from the cytosol(site of glycolysis ) into mitochondria(site of TCA
cycle)or is generated from alanine within mitochondria by transamination. Then pyruvate
carboxylase, a mitochondrial enzyme that requires coenzyme biotin as a carrier of activated HCO3-

,convertsthe pyruvate to oxaloacetate:

Because the mitochondrial membrane has no transporter for oxaloacetate, and therefore must be
reduced to malate by mitochondrial malate dehydrogenase, at the expense of NADH:

Malate leaves the mitochondria through a specific transporter(malate aspartate shuttle) in the inner
mitochondria membrane, and in the cytosol it reoxidized to oxaloacetate with the production of
cytosolic NADH.

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 The oxaloacetate is then converted to Phosphoenol pyruvate by phosphoenolpyruvate
carboxykinase

Conversion of Fructose 1,6-Bisphosphate to fructose 6-phosphate: Another reaction that cannot participate
in gluconeogenesis is the phosphorylation of fructose 6-phosphate by PFK-1. Because this reaction is highly
exergonic . In gluconeogenesis, generation of fructose 6-phosphate from fructose 1,6-bisphosphate is
catalyzed by a different enzyme fructose1,6-bisphosphatase(FBpase-1) through the addition of water
molecule and elimination of inorganic phosphate group(Pi).

2. Conversion of Glucose 6-phosphate to Glucose : The third bypass is the final reaction of
gluconeogenesis(dephosphorylation of glucose 6-phosphate to glucose). Reversal of this reaction is
energetically unfavorable. Therefore another enzyme glucose 6-phosphatase catalyzes the reaction
through a simple hydrolysis of phosphate ester and does not require synthesis of ATP.

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 Muscle and brain lack this enzyme and so cannot carry out gluconeogenesis . Glucose produced by
gluconeogenesis in liver or kidney or ingested in the diet is delivered to muscle and brain through the.
bloodstream.
Regulation of gluconeogenesis
1. Pyruvate Carboxylase
It is an allosteric enzyme. Acetyl CoA is an activator of pyruvate carboxylase so that generation of oxaloacetate
is favoured when the acetyl CoA level is sufficiently high.
2. Hormonal Regulation of Gluconeogenesis
The hormones glucagon and glucocorticoids and insulin regulate gluconeogenesis .
Glucagon favours gluconeogenesis by the production of glucose from Its stored form(glycogen) which
prevents blood sugar from falling and thus favours gluconeogenesis and inhibit glycogen Synthesis
(glycogenesis) and glycolysis
Glucocorticoids induce the synthesis of hepatic amino transferases thereby providing substrate for
gluconeogenesis.
Insulin inhibits the process of gluconeogenesis; by reducing the blood sugar(glucose);glucose synthesized in
gluconeogenesis is reduced which favours glycogenesis and inhibits gluconeogenesis.
3. ATP availability
Increased ATP(adenosine triphosphate) favours gluconeogenesis and vice-versa.
4. Fructose 1,6-bisphosphatase
The availability of the enzyme Fructose 1,6-bisphosphatase that catalyzes the conversion of fructose 1,6
phosphate to fructose 6-phosphate regulate gluconeogenesis. Increased in the enzyme favours
gluconeogenesis and vice versa.
Fructose 2,6-bisphosphateand AMPreduces the action of fructose 1,6-bisphosphate and increases the activity
of phosphofructokinase(glycolytic enzyme)

Relationship between Glycolysis and gluconeogenesis

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 Past questions on gluconeogenesis

• Explain the importance of glyoxylate pathway in carbohydrate metabolism

• State the roles of gluconeogenesis in man
• Write a concise note on gluconeogenesis
• Discuss the importance of Phosphoenol pyruvate and oxaloacetate in gluconeogenesis

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 • Discuss the reactions unique to gluconeogenesis.
• Enumerate the regulation of gluconeogenesis
• Briefly discuss the bypass reactions of gluconeogenesis.
• Explain why glucose cannot be formed from acetyl-CoA

Mighty T
Other Monosaccharides Enter the Glycolytic Pathway at several points

Fructose metabolism
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1. Kidney and muscle
D-fructose(ketohexose) present in free form in many fruits and formed by hydrolysis of sucrose in the
small intestine of vertebrates is phosphorylated by hexokinase.

The fructose-6-phosphate formed is converted to its isomer glucose-6-phosphate which later

continue the Emden Meyerhof pathway.

2. Liver
The liver enzyme fructokinase catalyzes the phosphorylation of fructose at C-1 rather than C-6 to
form fructose-1-phosphate.

The fructose-1-phosphate is then cleaved to glyceraldehyde and dihydroxyacetone phosphate by

fructose-1-phosphate aldolase(aldolase B).
Glyceraldehyde is phosphorylated by ATP and triose kinase to glyceraldehyde-3-phosphate.
Dihydroxyacetone phosphate is converted to glyceraldehyde-3-phosphate by the glycolytic enzyme
triose phosphate isomerase.

Defects of fructose metabolism

• Fructosuria
This is a metabolic defect due to the deficiency of the enzyme hepatic fructokinase.
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 Fructose is not converted to fructose-1-phosphate, thereby leading to the accumulation of
fructose; the fructose is excreted through urine.
Urine gives positive to benedict’s and Seliwanoff’s tests.
Treatment: Treatment of dietary fructose.

• Hereditary Fructose intolerance(HFI)

It is an autosomal recessive inborn error of metabolism. The defect is aldolase-B; hence
fructose-1-phosphate cannot be metabolised.
This is seen when sucrose(containing fructose) is introduced in the diet of infants, usually
around 6months of age.
Accumulation of Frutose-1-phosphate will inhibit glycogen phosphorylase. This leads to
accumulation of glycogen in liver and associated hypoglycemia.
Fructose is also excreted in urine when urine gives positive Benedict’s test.
Withdrawal of fructose from the diet will immediately relieve the symptoms

Galactose metabolism
D-galactose, a product of hydrolysis of the disaccharide lactose(milk sugar), passes in the blood
from the intestine to the liver, where it is first phosphorylated at C-1, at the expense of ATP,
by the enzyme galactokinase.

The galactose-1-phosphate is then converted to its epimer at C-4, glucose-1-phosphate, by a

set of reactions in which uridine diphosphate(UDP) functions as a coenzyme-like Carrier of
hexose groups.
Disease of galactose metabolism
• Galactosemia
Defects of any of the three enzymes in this pathway causa galactosemia inhumans.
In galactokinase deficiency galactosemia, there is:
High galactose concentrations are found in blood and urine

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 Infants develop cataracts, caused by deposition of the galactose metabolite(galactitol)in the
Lens.
In Transferase deficiency galactosemia; the effects is more severe;
Poor growth in children, speech abnormality, mental deficiency and liver damage that may be
fatal even when galactose is withheld from the diet.
Epimerase-deficiency galactosemia
It has a similar symptoms with transferase-deficiency but it is less severe when dietary
galactose is carefully controlled.

Mannose metabolism
D-mannose, released in the digestion of various polysaccharides and glycoproteins of foods,

can be phosphorylated at C-6 by hexokinase.

Mannose-6-phosphate is isomerized by phosphomannoseisomerase to yield fructose-6-

phosphate,an intermediate of glycolysis.

Past Questions
• Describe the pathway involved in the metabolism of galactose to glucose.
• Write comprehensively on the following terms: I. Galactosemia II. Fructosuria
• Draw the metabolic pathway of fructose and related disease conditions.
• Discuss the interplay between the metabolism of fructose and galactose

Mighty T

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 The Metabolism of Glycogen
(Glycogenesis and Glycogenolysis)
The synthesis of glycogen from glucose is referred to as glycogenesis. It takes place in all animal tissues but
chiefly occurs in the liver and skeletal muscle. Under well fed condition, about 245gms of glycogen is
stored in the muscle and 72gms of it in the liver. The main function of glycogen is to provide glucose during
starvation, and it also serves in:
• Maintaining blood glucose level.
• Acts as reserve fuel for muscle contraction.
• Storage form of carbohydrates in human body.

Glycogen as a storage form of glucose

Glycogen which is the polymeric form of glucose in animals in alpha-(1,4–1,6) glycosidic bond is considered
as the best storage of glucose due to:
• Glycogen is insoluble and it will not exact osmotic pressure
• Glycogen has high energy level than glucose
• Glycogen can be easily broken down into glucose
Also, the stored form of glucose cannot be fat because;
• Fat cannot be rapidly transported like that of glycogen
• Fat cannot be metabolised anaerobically
• In animals, fat cannot be converted to glucose due to the irreversibility in the reaction .

Reactions of the synthetic pathway

1. Activation of glucose
In this reaction, hexokinase(glucokinase) catalyzed the activation of glucose at C-6 using ATP as the
phosphoryl donor in the presence of Mg2+, which later form glucose-6-phosphate.

2. Mutation
The enzyme phosphoglucomutase catalyzed the conversion of glucose-6-phosphate to glucose-1-
phosphate. The reaction involves the phosphoryl shift from C-6 to C-1 of glucose.

3. Activation of glucose-1-phosphate
glucose-1-phosphate reacts with UTP(uridine triphosphate) to form active nucleotide UDP-
glucose(uridine diphosphate glucose) by the enzyme UDP-glucose phosphorylase.

4. Action of glycogen synthase

The glucose moiety from UDP-glucose is transferred to a glycogen primer(glycogenin) molecule. The
primer is essential as the acceptor of the glycosyl unit.
5. Introduction of Branching
The enzyme glycogen synthase can only add glucose units only in the alpha-1,4 linkage, and therefore
a branching enzyme is needed to create the alpha-1,6 linkages.
• When the chains is lengthened to 11-12glucose residues, the glycogen branching
enzyme(glycosyl (4-6) transferase) catalyzes the transfer of a terminal fragment of 6 to 7
glucose residues from the non-reducing end of a glycogen branch having at least 11residues to
the C-6 hydroxyl group of a glucose residue at a more interior position of the same or another
glycogen chain, thus creating a new branch.
To this newly created branch, further glucose units can be added in alpha-1,4 linkage by glycogen
synthase.

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 Regulation of glycogenesis
• Availability of substrate: In a well-fed state, when the blood glucose level is high, glucose-
6-phosphate, the substrate for UDP-glucose is high, which allosterically increases
glycogenesis. Also, during fasting, the substrate is low and there is need for glucose which
causes Glycogenolysis whichis opposite of glycogenesis.
• Hormone:
Glucagon and epinephrine are diabetogenic(increase blood glucose level) and thus antagonize
glycogen synthesis
Insulin which is an anti-diabetic hormone favours glycogenesis by lowering the blood
glucose level and storing it as glycogen .

Degradation of Glycogen(Glycogenolysis)
It Requires (3) enzymes;
a. Glycogen phosphorylase b. Glycogen debranching enzyme c. Phosphoglucomutase
1. Glycogen phosphorylase removes glucose as glucose-1-phosphate from glycogen . The alpha-
1,4linkages in the glycogen are cleaved. The enzyme sequentially hydrolyses alpha-1,4-glycosidic
linkages till it reaches a glucose residue. It cannot attack the 1,6linkage at branch point.
2. Debranching by bifunctional(two) enzymes
This enzyme removes the glycogen branches thereby making glycogen available for accessibility. A
block of 3 glucose residues are transferred from the branching point to another branch. This enzyme
is alpha-1,4-alpha1,4 glucan transferase. Now the branching is free. Then alpha-1,6-
glucosidase(debranching enzyme) canhydrolyse the remaining glucosyl unit held in alpha-1,6
linkage at the branch point. This glucose residue is released as free glucose.
3. Phosphoglucomutase
Phosphorylase reaction produces glucose-1-phosphate while debranching enzyme releases glucose.
The glucose-1-phosphate is converted to glucose-6-phosphate by phosphoglucomutase
4. Glucose-6-phosphatase in liver
Next, hepatic glucose-6-phosphatase hydrolyses glucose-6-phosphate to glucose. The free glucose is
released to the bloodstream.
Glycogen storage disease
1. Type 1(glucose-6-phosphatase deficiency) Von Gierke’s disease: The deficiency of this enzyme
leads to high accumulation of glycogen in the kidney and liver, this causes hypoglycemia(low
glucose level), hepatomegaly(enlarged liver).
2. Type II(alpha-1,4 glucosidase deficiency) pompe’s disease:This is the most devastating of the
glycogen storage diseases. It results in a large accumulation of glycogen in the lysosomes of all
cells which can lead to death by cardiorespiratory failure. Alpha-1,4 glucosidase is not involved in
the main glycogen metabolism. It occur in the lysosome, where it hydrolyse maltose and other
linear oligosaccharides thereby yielding glucose.
3. Type Vi(Liver phosphorylase deficiency) Hers disease: patient with a deficiency of liver
phosphorylase have symptoms related to type 1, in which the liver is unable to respond to
immediate glucose need (hypoglycemia)
4. Type 0(Liver glycogen synthase deficiency): This is the only type of glycogen metabolism
disease in which there is low glycogen present rather than overabundance of glycogen. This is due
to the low activity of glycogen synthase. It causes hyperglycemia.

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Mighty T
The Citric acid CYCLE
The complete cycle was proposed bySir Hans Krebsin 1937(Nobel prize, 1953). The cycle is therefore named
after him(Krebs cycle) afterward he proposed original name as TCA(tricarboxylic acid) cycle. Due to the
presence of citric acid as a member of the cycle, so the name Citric acid cycle was then given
Acetyl coA enters the cycle, and is completely oxidized. During this process, energy is trapped. Pyruvate
derived from glycolysis is oxidatively decarboxylated to acetyl coA by the pyruvate dehydrogenase. This is

the link between TCA cycle and glycolysis. The pyruvate dehydrogenation reaction occurs in the
mitochondria. Pyruvate with the help of a carrier can enter the mitochondria from the cytoplasm. The acetyl
coA derived from beta oxidation is formed in the mitochondria itself. All the enzymes of citric acid cycle are
located inside the mitochondria .
Sources of acetyl coA in Krebs cycle
• Glucose (Glycolysis)
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• Fatty acids(B-oxidation)
• Ketogenic amino acids (transamination reaction)
•
Reaction Steps of the Citric acid cycle

1. Formation of Citrate: The first reaction of the cycle is the condensation of acetyl-coA with
Oxaloacetate to form Citrate catalyzed by Citrate synthase.
In this reaction the methyl carbon of the acetyl group Is Joined to the carbonyl group(C-2) of
oxaloacetate leading to the removal of CoA and addition of Water molecule.

2. Formation of isocitrate via cis-Aconitate: The enzyme aconitase(aconitate hydratase)catalyzesthe

reversible transformation of citrate to isocitrate(isomer of citrate) through the intermediary
formation of the tricarboxylic acid cis-aconitate, which normally does not dissociate from the active
site. Aconitase contains an iron-sulfur center which acts both in the catalytic addition or removal of
water leading to the formation of either citrate and isocitrate.

3. Oxidation of isocitrate to a-ketoglutarate: In this reaction, the enzyme isocitrate dehydrogenase

catalyzes oxidative decarboxylation of isocitrate to form a-ketoglutarate. This reaction is involved
with the process of decarboxylation and dehydrogenation at C-2 and C-3 of isocitrate respectively.

4. Oxidation of a-ketoglutarate to Succinyl-coA: The next step is another Oxidative decarboxylation,

inwhich a-ketoglutarate is converted to succinyl-coA and CO2 by the action of the a-ketoglutarate
dehydrogenase complex; NAD+ serves as the Electron acceptor and CoA as the Carrier of the
succinyl group.

5. Conversion of Succinyl-coA to Succinate: The enzyme succinyl-coA synthetase(succinic

thiokinase) catalyzes the reversible reaction in the conversion of succinyl-coA to succinate in which
the phosphoryl group replaces the CoA of succinyl-coA bound to the enzyme Forming a High energy
acyl phosphate, which leads to succinate.

6. Oxidation of succinate to fumarate: The dehydrogenation(Oxidation) process involved in the

conversion of succinate to fumarate(the trans isomer of maleate)is catalyzed by the enzyme
Succinate dehydrogenase, aided by the coenzyme FAD as the electron acceptor forming its reduced
form(FADH2).

7. Hydration of Fumarate to Malate: The reversible hydration(addition of Water molecule)of fumarate

to L-malate is catalyzed by fumarase(fumarate hydratase).

8. Oxidation of Malate to Oxaloacetate: In the Last reaction of the Citric acid cycle, NAD -linked L-
malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate:
Regulation of the Citric acid cycle
1. Citrate and Citrate synthase: The formation of citrate from oxaloacetate and acetyl coA is as
important part of control. ATP acts as an allosteric inhibitor of citrate synthase. Citrate
allosterically inhibits PFK, the key enzyme of glycolysis; stimulates fructose-1,6-bisphosphatase, a
key enzyme of gluconeogenesis and activates acetyl coA carboxylase, key enzyme of fatty acid
synthesis

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 2. Availability and cellular need of ATP :when the energy charge of the cell is low, as indicated by
high level of NAD+ and FAD, the cycle operates at faster rate and vice versa. The Krebs cycle is the
largest generator of ATP among metabolic pathways.
3. Isocitrate Dehydrogenase : In the step 3 of TCA cycle, ADP acts as a positive modifier enhancing
the binding of substrate. NADH is as inhibitor.
4. Alpha ketoglutarate dehydrogenase :It is inhibited by succinyl CoA and NADH.
Importance of the Citric acid cycle
1. It is the largest generator of ATP among metabolic pathways
2. It is the Source of reduced co-enzyme (NADH, FADH2) that provide the substrate for the respiratory
chain
3. The cycle plays an amphibolicrole(link between catabolic and anabolic pathways)
4. It provides precursors for synthesis of amino acids and nucleotides
5. Generationof oxaloacetate and malate in the first bypass reaction(conversion of pyruvate to
Phosphoenol pyruvate) of gluconeogenesis

Energy Yield(number of ATP generated) in Krebs cycle

Step Enzyme Source No. of ATPs generated

3 Isocitrate NADH 3*1=3 (2.5*1=2.5)

dehydrogenase

4 a-Ketoglutarate NADH 3*1=3(2.5*1=2.5)

dehydrogenase

5 Succinyl-coA GTP 1*1=1

Synthetase/succinate
thiokinase

6 Succinate FADH2 2*1=2 (1.5*1=1.5)

dehydrogenase

8 Malate dehydrogenase NADH 3*1=3 (2.5*1=2.5)

Total ATP=12or 10
Note:
• Krebs cycle is also called Tricarboxylic cycle(TCA cycle)due to the presence of three carboxylic
group in the structure of tricarboxylic acid(citric acid) which is a member of the cycle.
• The reaction take place in the Mitochondria of cells.
• All reactions in this cycle are reversible with the exception of reaction 1,3 and 4.
• Co-enzyme (NADH) is located at reaction 3,4 and 8;
• (FADH) at reaction 6; (GTP) at reaction 5.
• The members of the cycle can be formulated as (At comedy international, Alfa Sule Search for
more offer)
• NADH =3 ATP / 2.5 ATP and FADH=2 ATP / 1.5 ATP
• The citric acid cycle has eight steps
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 • The main function of this cycle is Energy production(ATP)
• A total of 12ATP is generated per cycle
Past questions on Krebs cycle
• Enumerate the energy produced by the TCA(Krebs) cycle
• Write a comprehensive note on tricarboxylic acid cycle
• Discuss the importance and regulation of TCA cycle
MightyT
Pentose phosphate/ Phosphogluconic pathway of Glucose oxidation
This is another fundamental important pathway of Glucose 6-phosphate Catabolism(the major catabolic fate
is Glycolysis) that take place in rapidly dividing cells such as those of bone marrow, skin and intestinal
mucosa. It is called hexose monophosphate pathway. The cell uses pentoses to make RNA, DNA and
coenzymes such as ATP, NADH, FADH2 and coenzyme A. Unlike glycolysis, the phosphogluconate
pathway does not involve production of ATP but essential NADPH(reducing power).
Pentose phosphate pathway(PPP) has two phases:
• Oxidative phase (Glucose 6-phosphate is degraded to yield pentose phosphate and NADPH)
• Non-oxidative phase (Pentose phosphate is recycled to Glucose 6-phosphate

The oxidative phase reactions( production of Pentose Phosphates and NADH)

1. Oxidation of glucose 6-phosphate: The first reaction of the pentose phosphate pathway is the
oxidation of glucose 6-phosphate by glucose 6-phosphate dehydrogenase(G6PD)to form 6-
phosphoglucono-d-lactone, an intramolecular ester with NADP+ as the electron acceptor forming
NADPH.

2. Hydration of lactone : The Phosphoglucono-d-lactone is hydrolysed to the free acid 6-phospho-

gluconate catalyzed by a specific enzyme lactonase.

3. Oxidative decarboxylation of 6-phosphogluconate: The enzyme 6-phosphogluconate

dehydrogenase catalyzes the oxidation and decarboxylation of 6-phosphogluconate to form
ketopentose ribulose5-phosphate. This reaction generates a second molecule of NAD

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 4. Conversion of ribulose 5-phosphate to ribose 5-phosphate: In this reaction, Phosphopentose.
isomerase converts ribulose5-phosphate to its aldose isomer, ribose 5-phosphate. The reaction is
termed isomerization reaction.
Note: In some tissues, the pentose phosphate pathway ends at this point

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The Nonoxidative phase reactions(recycles pentose phosphates to Glucose 6-phosphate)

5. Conversion of ribulose 5-phosphate to xylulose 5-phosphate : In the first reaction of non-oxidative
phase, ribose5-phosphate epimerase catalyzes the conversion of ribulose 5-phosphate(a member of
the oxidative phase) to its epimer xylulose 5-phosphate .

6. Action of Transketolase: One of the two enzymes unique to the phosphogluconate pathway is
Transketolase (an enzyme that transfer two carbon unit)which transfers the C-1 and C-2 of xylulose
5-phosphate to ribose 5-phosphate forming the seven-carbon product sedoheptulose 7-phosphate.
The remaining three-carbon fragment from xylulose is glyceraldehyde 3-phosphate.

7. Action of Transaldolase: The other unique enzyme in phosphogluconic pathway is Transaldolase

which catalyzes the transfer of a three carbon fragment from sedoheptulose 7-phosphate and
condenses with glyceraldehyde 3-phosphate, forming fructose 6-phosphate and the tetrose
erythrose 4-phosphate .

8. Action of Transketolase catalyzes the transfer of two carbon unit(C-1 and C-2) from a ketose donor
xylulose 5-phosphate to an aldose acceptor erythrose 4-phosphate, forming fructose 6-phosphate
and glyceraldehyde 3-phosphate.
• Note: The action of transketolase is similar to that of transaldolase, but still differ;
Transketolase : catalyzes the transfer of two carbon unit.
Transaldolase : catalyzes the transfer of three carbon unit.

9. Regeneration of glucose 6-phosphate: Two molecules of glyceraldehyde 3-phosphate formed from

these reaction can converted to glucose 6-phosphate(a precursorof oxidative phase of pentose
phosphate pathway)through the process of gluconeogenesis. Also, the fructose 6-phosphate produced
serves as an intermediate in glycolysis and gluconeogenesis.

Regulation of HMP shunt pathway

• Availability of coenzyme
The pathway is mainly regulated by the level of NADP+. The first reaction catalyzed by Glucose 6-phosphate
dehydrogenase is the rate-limiting step and it is inhibited by NADPH. The oxidative phase is therefore

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controlled by the level of NADP+ and non-oxidative phase by the requirement of pentoses. When more
NADPH is needed, the pathway increases and vice versa.
• Hormonal action
The action of insulin induce glucose 6-phosphate dehydrogenase and therefore will increase the overall
pathway.

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Physiological Importance of pentose phosphate pathway / NADH
1. It serves as another catabolic fate of glucose 6-phosphate which lead production of specialized products
such as Ribose 5-Phosphate, NADPH needed by the cell
2. Rapidly dividing cells(bone marrow, skin, and intestinal mucosa, use the pentoses(Ribose 5-
phosphate) as a precursor in purine biosynthesis in RNA and DNA production.
3. In other tissues, the essential product of pentose phosphate pathway is not the pentoses but electron
donor NADPH, needed for reductive biosynthesis.
4. The NADPH is also used to counter the damaging effects of oxygen radicals.
5. Pentose phosphate pathway helps in the generation of NADPH for tissues that carry out extensive fatty
acid synthesis (liver, adipose, Lactating mammary gland) and cholesterol and steroid biosynthesis
(liver,adrenal gland, gonads)
6. The generated NADPH is used in preventing oxidative damage caused by the deficiency of glucose 6-
phosphate dehydrogenase(the first enzyme of the pathway)
7. Production of glyceraldehyde 3-phosphate for conversion to fructose 1,6-bisphosphate in
gluconeogenesis and production of glycolytic intermediates
8. In human, NADPH is needed for the normal maintenance of Red blood cell where it serve to generate
reduced Glutathione(GSH), a deficiency of which result in haemolysis of the cell.
9. NADPH is required for preserving the transparency of lens.
10. Most of the drugs and other foreign substances are detoxified by the liver microsomal P450 enzymes,
with the help of NADPH.

Past questions on pentose phosphate pathway .

• State the importance of hexose monophosphate shunt in man
• State the importance of the NADPH and ribose-5-phosphate in pentose phosphate pathway.
• Highlight differences between the two phases of the hexose monophosphate shunt.
• Write an essay on the metabolism and importance of pentose phosphate shunt.
• Write a comprehensive essay on pentose phosphate pathway.

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Mighty T
Biosynthesis of Triglycerides and Phospholipids

Biosynthesis of Triglycerides (Triacylglycerol)

Humans can store only a few hundred grams of glycogen in liver and muscle, barely enough to supply the
body for 12hours. Whenever carbohydrates is ingested in excess of the organisms capacity to store glycogen,
the excess glycogen is converted to TAG and stored in adipose tissue.
In a man weighing 70kg, the total TAG found is 15kg which can support basal energy for 12weeks.
TAG in plants are mainly stored in fruits, nut and seeds.
When the diet contains more fatty acids than are needed immediately as fuel, they are converted to TAG in
the liver and packaged with specific apolipoprotein into very low density lipoprotein(VLDL).

Site of TAG biosynthesis: Liver and adipose tissues are the major sites.
The starting material in TAG biosynthesis is Sn glycerol-3-Phosphate.

Precursors(Sources) Of glycerol-3-Phosphate
1. Phosphorylation of glycerol: The enzyme glycerol kinase catalyzed the transfer of a phosphoryl
group from a nucleoside triphosphate (ATP) to the C-3 of glycerol yielding glycerol-3-Phosphate.
This process does not occur in the adipose tissue due to the deficiency of glycerol-kinase. It occurs
in the liver.

2. Reduction of Dihydroxyacetone phosphate(DHAP): The glycolytic intermediate

Dihydroxyacetone phosphate which is one of the triose phosphate produced from the cleavage
of fructose 1,6 bisphosphate is reduced by the action of the Cytosolic NAD-linked glycerol-3-
phosphate dehydrogenase to yield glycerol-3-phosphate.
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 3. Denovo production by a process known as glyceroneogenesis via pyruvate: Glyceroneogenesis
is the shortened version of gluconeogenesis, from pyruvate to Dihydroxyacetone
phosphate(DHAP) followed by the conversion of DHAP to glycerol-3-phosphate by the action of
the Cytosolic NAD-linked glycerol-3-phosphate dehydrogenase.

Pyruvate—oxaloacetate—PEP—2PG—3PG—1,3BPG—G3P—DHAP—Glyerol-3-Phosphate.

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Process of TAG biosynthesis

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The first stage of biosynthesis of TAG is the acylation of the two free OH group of Sn glycerol-3-phosphate
by two molecules of fatty acyl-CoA to yield diacylglycerol-3-phosphate(Phosphatidic acid) which can be
converted to TAG or glycerolphospholipid.
Inthe pathway to TAGbiosynthesis, Phosphatidic acid is hydrolysed by phosphatidic acid phosphatase to
form 1,2 diacylglycerol, whichis later converted to TAG by transesterification through the enzyme(acyl
transferase) with a third fatty acyl-CoA.

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Reactions in the biosynthesis of glycerolphospholipid

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The first stage in the biosynthesis of glycerolphospholipid are shared with the pathway of Triacylglycerol,
which involves the acylation of the two free OH group of Sn glycerol-3-phosphate with two molecules of fatty
acyl CoA, a reaction catalyzed by acyl transferase to yield Diacylglycerol-3-Phosphate(Phosphatidic acid).
The diacylglycerol is activated by condensation with Cytidine triphosphate(CTP) to form CDP-
diacylglycerol, with the elimination of pyrophosphate. Displacement of Cytidine Monophosphate(CMP)
through the nucleophilic attack by the hydroxyl group of serine yields Phosphatidylserine(PS).
Decarboxylation of the serine moiety in PS catalyzed by phosphatidylserine decarboxylase yields
phosphatidylethanolamine(PE). PE may also be converted by the addition of 3-methyl groups to its amino
group.

Past questions on the biosynthesis of TAG and PPL

• List and discuss the three sources of glycerol-3-phosphate as required in triacylglycerol synthesis.
• Discuss the biosynthesis of phosphatidylcholine.
• Discuss the synthesis and the biochemical significance of triacylglycerol(TAG)
• Write short note on the following:
Phosphatidylserine
Phosphatidylethanolamine
Phosphatidylcholine.

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Mighty T
Cholesterol biosynthesis

Cholesterol is a precursor of steroid hormones; it is an essential molecule in many animals(humans),it is not

required in mammalian diet. The word Cholesterol is derived from Greek words: Chole– bile; Steros –solid;
Ol–alcohol.In a man weighing 70kg, a total of about 140g of cholesterol is found.

In 1940, Konrad Bloch discovered that all carbon atoms of cholesterol are derived from acetyl CoA(Nobel
price in 1964). Cholesterol 27C compound suggests a complex pathway. The biosynthetic pathway was
described by Sir John Cornforth and Vladimir Prelog(Nobel prizes,1975).

Site of cholesterol biosynthesis: livre, adrenal cortex, testis, Ovaries and intestine. All nucleated cells can
synthesize Cholesterol including arterial walls.
The enzyme involved in the synthesis of cholesterol are partly in the endoplasmic reticulum and partly in the
cytoplasm.
Stages of Cholesterol biosynthesis

1. Synthesis of Mevalonate from acetate.

2. Conversion of Mevalonate to Two activated isoprene.
3. Condensation of Six Activated isoprene units to form Squalene.
4. Conversion of squalene to Cholesterol.

1. Synthesis of Mevalonate from acetate :Condensation of acetyl CoA leads to the formation of
acetoacetyl CoA, which later condenses with the third acetyl CoA to form B-hydroxyl-B-methyl
glutaryl CoA(HMG CoA) , these reactions is acted upon by Thiolase and HMG-CoA synthase

respectively. The next reaction involves the donation of two electrons from NADPH to replace the acyl group
of the HMG CoA leading to the formation of the first intermediate of Cholesterol biosynthesis which is
Mevalonate. This reaction acted upon by HMG CoA reductase is the committed and rate limiting step.
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 2. Conversion of Mevalonate to two activated Isoprenes: Mevalonate is converted to two activated
isoprene by the addition of three phosphate groups from ATP molecules leading to the formation of
an intermediate; 3-phospho-5-pyrophosphate Mevalonate. The phosphate group at C-3 of the
compound is a leaving group.

The phosphate group and the carboxyl group leave, producing a double bond in the five carbon product;
Isopentenyl pyrophosphate, which is one of the two isoprene units of cholesterol biosynthesis. Isomerization
of Isopentenyl pyrophosphate gives dimethyl allyl pyrophosphate, the second isoprene units.

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 3. Condensation of 6 activated isoprene units to form squalene: Isopentenyl pyrophosphate and
dimethyl allyl pyrophosphate now undergo head-tail condensation to form geranyl pyrophosphate,
which is formed by the displacement of one of the pyrophosphate. Geranyl pyrophosphate also undergo
head-tail condensation with Isopentenyl pyrophosphate by the action of the enzyme prenyl transferase
leadingto the formation of Farnesyl pyrophosphate.

Two molecules of farnesyl pyrophosphate now undergoes head-head condensation to form Squalene.

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4. Conversion of Squalene to Cholesterol: The action of squalene monoxygenase addsone oxygen
atom of O2 to the end of the squalene chain, forming an epoxide. NADPH reduces the oxygen atom
of O2 to H2O. The double bonds of the product Squalene 2,3-epoxide are positioned so that a

remarkable concerted reaction can convert the linear Squalene epoxide to a cyclic structure. In animal
cells, this cyclization results in the formation of lanosterol, which contain the four rings characteristics
of the steroid nucleus. Lanosterol is finally converted to Cholesterol in a series of about 20reactions
that include the migration of some methyl groups and removal of others.
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 Fate of Cholesterol
• Degradation to CO2 : In human tissues, this conversion does not occur)
• Conversion to Bile acids: The major pathway, more than 50% is converted to bile acids and
excreted in faeces.
• Conversion to Neutral Sterols : 10% of cholesterol is converted to Coprosterol.
• Formation of cholesteryl ester through acyl CoA transferase(ACAT)
• Formation of steroid hormones (testosterone, progesterone)

Regulation of cholesterol biosynthesis

The regulatory enzyme is HMG CoA reductase.

1. Regulation at transcription: Long-term regulation involves the regulation at transcription of the gene
for HMG CoA reductase. When sufficient cholesterol is present in the cell, transcription of the gene
of HMG CoA reductase is suppressed, and cellular synthesis of cholesterol is decreased. When
cholesterol in diet is low, synthesis is increased.
2. Insulin and thyroxine increase the activity of HMG CoA reductase
3. Cortisol and glucagon decrease its activity
4. Drugs: Lovastatin and other statin group of drugs are competitive inhibitors of HMG CoA reductase.
So, they are used in clinical practice to reduce cholesterol level in blood.

Bile acid formation

Bile acids are synthesized in the liver from cholesterol. They contain 24 carbon atoms, and are relatively
hydrophilic and aid lipid digestion. All of them have an alpha-oriented hydroxyl group at position 7.
Types

1. Primary bile acid: They are synthesized in the liver from cholesterol. They are mainly two:
• Cholic acid(3,7,12-trihydroxycholanic acid) :Quantitatively the largest in amount
• Cheno-deoxycholic acid(3,7-dihydroxycholanic acid)
2. Secondary bile acid: They are produced in the intestine from primary bile acid by the action of
intestinal bacteria, they are produced by deconjugation. The deconjugated bile acids are then partly
converted to secondary bile acids by the removal alpha hydroxyl group at position 7. Cholic acid is
thus converted to deoxycholic acid and chenodeoxycholic acid to lithocholic acid.
• Deoxycholic acid formed from Cholic acid
• Lithocholic acid from chenodeoxycholic acid.
Role of bile acid
• Elimination of cholesterol from the body
• It increases the absorption of fats
• Bile acid-containing micelles aid lipases to digest lipids.
• Many waste products including bilirubin are eliminated from the body by secretion into bile
and elimination in faeces.

Past questions on cholesterol biosynthesis

• State the unique steps in cholesterol biosynthesis
• Briefly describe the rate limiting step in cholesterol biosynthesis
• Discuss the regulatory mechanisms in cholesterol biosynthesis
• Mention one bile acid and discuss its biosynthesis from a two-carbon compound
• Write the role of bile acid in the catabolism of fatty foods.
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 • Discuss the biosynthesis of a named bile acid from two carbon compound.
Mighty T

De novo synthesis of fatty acids

The process of fatty acid synthesis was studied by Feodor Lyen(Nobel prize, 1964). This pathway is also
referred to as Lynen’s spiral. The process occurs in liver, adipose tissue, kidney, brain and mammary
glands.
Site of reaction: The biosynthesis of fatty acid takes place in the cytoplasm.
The starting material for denovo synthesis of fatty acid is acetyl-CoA.
Note: Acetyl CoA is formed inside the mitochondria from pyruvate and therefore needs to be transported.

Transport of Acetyl CoA from mitochondria to cytoplasm

Pyruvate is converted to acetyl CoA in the mitochondria. The inner membrane is not freely permeable to
acetyl CoA. Hence, the acetyl CoA units is first converted to citrate and transported from the
mitochondria into the cytoplasm through a tricarboxylic transporter. In the cytoplasm, the citrate is then
cleaved to oxaloacetate and acetylCoA, catalysed by the action of ATP citrate lyase. The oxaloacetate then
return to the mitochondria as malate or pyruvate.

Stages of fatty acid biosynthesis

This biosynthetic system is catalysed by the action of a multi-enzyme complex known as Fatty acid
synthase(FAS) complex. The enzymes are grouped into three units;
• Condensing unit : It is the initial substrate binding site. The enzymes involved are acetyl
transacylase(AT), malonyl transacylase and beta-keto acyl synthase or condensing enzyme(CE)
catalysing reaction 2 and 3 respectively.
• Reduction unit: it contains keto acyl reductase, dehydratase, enoyl reductase and acyl carrier
protein(ACP) catalysing step 4,5,6 respectively
• Releasing unit: it is involved in the release of the palmitate synthesised. It contains thio-
esterase(TE) or deacylase,catalysing the last reaction.

Step 1: Carboxylation of acetyl CoA

The first step in the fatty acid synthesis is the carboxylation of acetyl CoA to form malonyl CoA catalysed
by acetyl CoA carboxylase which requires a coenzyme biotin(carrier of activated carboxyl group).This
enzyme is not a part of the multi-enzyme complex. But it is the rate-limiting enzyme.

Step 2: Binding of a molecule of acetyl CoA and malonyl CoA to a multi-enzyme complex
a. The acetyl transacylase(AT) catalyses the transfer of the acetyl group to the cysteinyl SH group of
the condensing enzyme(CE)
b. The Malonyl transacylase(MT) transfers the malonyl group to the SH group of the Acyl carrier
protein(ACP).

Step 3: Condensation
The acetyl(2c) and malonyl(3c) units are condensed to form beta-keto acyl ACP(acetoacetyl ACP). During
this process, one carbon is lost as Co2. The enzyme is called condensing enzyme(keto acyl synthase)

Step 4: Reduction
The acetoacetyl ACP is reduced by NADPH dependent beta-keto acyl reductase(KR) to form beta-
hydroxyl fatty acyl ACP.

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Mighty T

Step 5: Dehydration
The beta hydroxyl butyryl ACP is then dehydrated by a dehydratase(DH) to form enoyl ACP (alpha beta
unsaturated acyl ACP)

Step 6: Second reduction

The enoyl ACP is again reduced by enoyl reductase(ER) utilising another molecule of NADPH to form
butyryl ACP.

Cycling of Reactions
The butyryl group(4c) is now transferred to the SH group of the condensing enzyme and another malonyl
CoA to the SH group of Acyl carrier protein. The sequence of reactions namely condensation, dehydration
and reduction(steps 3,4,5,6) are repeated. The cycles are repeated a total of seven times, till the 16-
Carbon palmitic acid is formed.

Step 7: Palmitic acid is released

The thio-esterase(TE)or de-acylaseactivity releases palmitate from the multi-enzyme complex.
The end point is palmitic acid(16c) in the Liver and adipose tissue. But in the lactating mammary gland,
the end product are Capric(10C) and Lauric(12C). Cow milks contain odd numbered fatty acids.

Past Question on fatty acid biosynthesis and Beta-oxidation

• Write a concise note on the biosynthesis of fatty acid in mammals
• List all the enzymes in fatty acid biosynthesis and the reaction they catalyze.
• Give the total and net ATP generated from the oxidation of the following fatty acids:
16C,15C, 17C, and 24C fatty acids.
• Write comprehensively on the role of carnitine shuttle
• Discuss the palmitic acid degradation
• Write a comprehensive note on beta-oxidation of odd chain fatty acids
• Discuss the degradation of fatty acid
• Beta-oxidation of saturated fatty acid occurs in the mitochondria. Discuss
• Give an account of the total ATP generated from the total catabolism of a named saturated
fatty acid.
• How many molecules of ATP are generated when Arachidonic acid(C20) is degraded.

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Mighty T Fatty acid Beta-oxidation
This is the process by which fatty acids are broken down by various tissues to produce energy. It is referred to as beta-
oxidation due to the oxidation of fatty acid at the beta-carbon atoms starting from the carboxylic end of each fatty
acids.
Site of reaction: It occurs in the Mitochondrial matrix.
This process occurs in cycles repeatedly depending on the length of carbon atoms of each Fatty acids.
Beta-oxidation involves the splitting of fatty acyl molecules to a two carbon units(acetyl CoA). The acetyl CoA
produced is also determined by the carbon length of fatty acids.
Note: The oxidation of fatty acid in the mitochondria(site of beta-oxidation), occur only after its activation in the
cytoplasm(site of FA synthesis) by the action of co-enzymeA to form acyl CoA.
• Beta-oxidation helps in the release of chemical energy contained in Fatty acids
• It generates acetyl CoA for efficiency of Krebs cycle , which eventually leads to energy(ATP) generation.

Preparative steps for beta-oxidation

1. Activation of fatty acids

Fatty acids are activated to their Co-enzyme A(CoA) derivative(acyl-CoA) .This activation takes place in the
cytoplasm. ATP is hydrolysed to AMP and PPi. This reaction is catalyzed by the enzyme thiokinase or fatty
acyl CoA
synthetase.

2. Transport of activated fatty acid

Fatty acids are activated in the cytoplasm; but the beta-oxidation is in the mitochondria. So transport through
the mitochondrial matrix is essential. The long chain fatty acyl CoA cannot pass through the inner
mitochondrial membrane; therefore it binds to the hydroxyl group of carnitine.
Therefore requires a transporter Carnitine—(CH3)3—N+—CH2—CHOH—CH2—COOH.
Enzymes of Carnitine and their functions
• Carnitine Acyl transferase-I(CAT-I): It transfers the fatty acyl group which cannot cross the mitochondrial
membrane to the hydroxyl group of carnitine to form acyl carnitine. This reaction occur in the cytosolic side
of inner mitochondrial membrane .

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 • Carnitine acyl translocase: A protein translocase will carry the acyl carnitine across the membrane to the
matrix of mitochondria.
• Carnitine acyl transferase II(CAT-II): On the matrix side of mitochondrial, CAT-II transfer the acyl group back
to co-enzyme A and the carnitine returns to the Cytosol by the action of translocase.

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Reactions of Beta-oxidation

Beta oxidation involves four major reactions: a. Oxidation b. Hydration c. Oxidation d. Cleavage

1. Oxidation: The fatty acyl CoA is dehydrogenated by the action of Acyl CoA dehydrogenase to a trans enoyl
CoA with FAD accepting the hydrogen atoms. FADH2when oxidised in electron transport chain will produce
2ATP molecules.

2. Hydration: This reaction is catalysed by enoyl CoA hydratase, which involves the addition of H20 to enoyl CoA
to form beta-keto fatty acyl CoA.

3. Oxidation: The beta-hydroxyl acyl CoA is oxidised again by the action beta-hydroxyl acyl CoA dehydrogenase
to form beta-keto acyl CoA, with the production of NADH.

39
 4. Cleavage: The beta-keto acyl CoA now undergoes thiolytic cleavage, splitting off a molecule of acetyl CoA and
leaving behind a fatty acid with a 2 carbon atoms less. This reaction is catalysed by Thiolase or acyl CoA acetyl
transferase.
Note: The newly formed fatty acyl CoA will sequentially undergo further cycles of steps 1,2,3 and 4 of B-
oxidation until Fatty acid is completely converted to acetyl CoA.

Energy derivation in the complete oxidation saturated fatty acids in Beta-Oxidation and Krebs cycle

• Energy yield in Beta-oxidation(per cycle)

Reaction 1—Acyl CoA dehydrogenase —1FADH2—2ATP
Reaction 3—Beta-hydroxyl acyl CoA DH—1NADH—3ATP
Energy yield in B-oxidation =5ATP
• Energy yield in Krebs cycle (Per acetyl CoA yield)
Reaction 3—Isocitrate Dehydrogenase—1NADH=3ATP
Reaction 4—alpha-ketoglutarate DH— 1NADH=3ATP
Reaction 5—Succinyl-CoA synthetase —1GTP=1ATP
Reaction 6—Succinate dehydrogenase—1FADH2=2ATP
Reaction 8—Malate dehydrogenase —1NADH=3ATP
Energy yield in TCA cycle =12ATP
Note: The Last acetyl CoA group in the Last cycle of FA does not undergo beta-oxidation but it is counted with the
acetyl coA produced. Therefore in beta-oxidation, the acetyl CoA produced is one greater than the cycle produced.
No of acetyl CoA =Number of carbon. No of cycle =No of acetyl CoA—1
2
Examples: Calculate the ATP generated from the complete oxidation of (a) Palmitic acid (b) stearic acid
Solution
Palmitic acid=16carbon(it undergoes 7cycles of B-oxidation and produces 8acetyl CoA)
• 8acetyl CoA × 12ATP(Krebs cycle) =96ATP
• 7FADH2 × 2ATP(step 1) =14ATP(Beta-oxidation)
• 7NADH ×3ATP(step 3) =21ATP(Beta-oxidation)
Gross total. =131ATP
Net yield. =131-2ATP for activation =129ATP
Stearic acid=18carbon unit(it undergoes 8cycles of B-oxidation and produces 9acetyl CoA)
• 9acetyl CoA × 12ATP. =108ATP
• 8FADH2 × 2ATP. =16ATP
• 8NADH × 3ATP. =24ATP
Gross total. =148ATP
Net yield. =148-2 =146ATP

Oxidation of odd chain fatty acids

The oxidation of odd chain fatty acids only differ from even chain oxidation at the last cycle, which produces a
Propionyl CoA(3c) instead of acetyl CoA(2c) . The Propionyl CoA is then converted to Succinyl-CoA, which later get
converted to acetyl CoA(2C) and finally enters the TCA cycle.
Stages of conversion
• Propionyl CoA —Succinyl CoA = -1ATP
• Succinyl CoA —Succinate; GTP=1ATP
• Succinate — Fumarate; FADH2 =2ATP
• Pyruvate —Acetyl CoA; NADH = 3ATP
• Acetyl CoA —TCA cycle. =12ATP
Energy yield from Propionyl conversion=18ATP-1ATP=17ATP
Note: Odd chain fA oxidation yields equal Number of cycle and acetyl CoA with one Propionyl CoA
Example 2: Calculate the number of ATP generated in 25c fatty acids
Number of Cycle =Number of acetyl CoA = N-3. ; (when n =25) = 25-3. = 22. =11cycles and 11acetylCoA

40
 2 2 2
Therefore a 25c produces 11cycles,11acetyl CoA and a Propionyl CoA
• 11acetyl CoA × 12ATP =131ATP
• 11FADH2 × 2ATP. =22ATP
• 11NADH × 3ATP. =33ATP
• 1propionyl CoA. =17ATP
Total energy yield= 204ATP-2ATP for activation = 202ATP
Plasma protein
Total plasma is 400-600mg/dl containing one-third of cholesterol, triglyceride, phospholipids. Since lipids
are insoluble in water, they need of carriers in plasma. They are complexed with proteins to form lipoproteins.
The protein part of lipoprotein(Lp) is called apolipoprotein.
Classification of Lipoproteins
Lipoproteins are classified into five(5) major types depending on the density by :
• Ultrafiltration
• Electrophoretic mobility

1. Chylomicrons (The protein attaching is apo-B-48)

2. Very low density lipoprotein(VLDL) Or pre-beta lipoproteins
3. Intermediate density lipoprotein(IDL) or broad-beta lipoprotein
4. Low-density lipoprotein(LDL) or beta-lipoproteins
5. High density lipoproteins(HDL) or alpha-lipoproteins

General Characteristics of Lipoproteins

• The lipoprotein molecules have polar peripheral made up of protein and polar heads of phospholipids
and cholesterol
• The inner core consists of hydrophobic TAGs and the tails of phospholipids.
• Apoprotein increase the solubility of lipoprotein
• Separation by centrifugation: Lipoprotein with High lipid will have a low density and so Will float
on centrifugation while the low lipid content lipoprotein will sediment and have high density.
• Separation by Electrophoresis: lipoproteins with higher protein content will move faster towards the
anode and those with less proteins have minimum mobility .

Apolipoprotein(protein portion)
The protein part of lipoprotein is called apolipoprotein(apo-Lp) or apoprotein. All apoproteins are mainly
synthesized in the liver; but small quantities are produced from almost all organs. Intestinal cells produce
small quantities of apo.
Functions of apoproteins
1. Apo-A-I: it activates lecithin-cholesterol acyl transferase(LCAT). It is the ligand for HDL receptor. It
is anti-atherogenic.
2. Apo-B-100: it is the only apoprotein in LDL; it binds to LDL receptor on tissues.
3. Apo-B-48: it is the apoprotein of chylomicrons. Apo-B-100 and Apo-B-48 are products of the same
gene. B-48 is so named because it is only 48% of the size of B-100.
4. Apo-C-ii: It activates lipoprotein lipase.
5. Apo-E: it is a universal lipoprotein, an arginine rich protein. It is present in chylomicrons, LDL and
VLDL. It is involved in the cellular transport of lipid.
Features of lipoprotein
1. Chylomicrons
They are the transport form of dietary triglycerides from intestines to the adipose tissues for
storage; and to muscle or heart for their energy needs.
2. VLDL

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 VLDL carries triglycerides(endogenous triglycerides) from the liver to peripheral tissues for
energy needs.
3. LDL
It transports cholesterol from liver to the peripheral tissues.LDL concentration in blood has
positive correlation with incidence of cardiovascular diseases. It is also referred to as bad
cholesterol because about 75% of the plasma cholesterol is incorporated into the LDL
particles.
LDL increase cholesterol content and thus atherogenic.
4. HDL
It is called good cholesterol because it serves as the main transport form of cholesterol from
peripheral tissues to liver, which is excreted through bile. This is called reverse cholesterol
transport by HDL.
The only excretory route of cholesterol from the body is bile.
HDL is anti-atherogenic .

Lipid metabolism within the adipose tissue

The adipose tissue serves as a storage of excess calories ingested. The triglycerides stored in the adipose tissue
are not inert. They undergo a daily turnover with new triacylglycerol molecules being synthesized and a
definite fraction being broken down.

• Adipose tissue in a well-fed condition

There is active lipogenesis(synthesis of lipid) in the adipose tissue.
The dietary triglycerides transported by chylomicrons and the endogenously synthesized triglycerides from
liver brought by VLDL are both taken up by adipose tissue and esterified and stored as TAG.
In a well fed condition, glucose and insulin levels are increased. The High level of Insulin causes :
1. Increased activity of glycolytic enzymes
2. Increase in pyruvate dehydrogenase activity
3. Increase in activity of acetyl CoA carboxylase
4. Increase in activity of glycerol phosphate acyl transferase
5. It inhibit hormone sensitive lipase

• Adipose tissue in a fasting condition

The metabolic pattern totally changes under conditions of fasting. TAG from the adipose tissue is mobilized
under the effect of the hormones, glucagon and epinephrine.
Under starvation, a high glucagon, ACTH, glucocorticoids and thyroxine have lipolytic effect. The released
free fatty acids (FFA) are taken up by peripheral tissues as a fuel.

• Adipose tissue and Diabetes mellitus

Lipolysis isenhanced and high FFA level in plasma is noticed in diabetes mellitus. The insulin is acting through
receptors on the cell surface of adipocytes. These receptors are decreased, leading to insulin insensitivity in
diabetes, when there is no insulin, lipolysis will be enhanced.

• Adipose tissue and Obesity

The fat content of the adipose tissue can increase to unlimited amounts, depending on the amount of excess
calories taken in. This leads to Obesity. High levels of plasma insulin level is noticed. But the insulin
receptors are decreased; and there is peripheral resistance against insulin action. When fat droplets are
overloaded, the nucleus of adipose tissue cell is degraded, cell is destroyed, and TAG becomes extracellular.
Such TAG cannot be metabolically reutilized and forms the dead bulk in obese individuals.

Fatty Liver
Fatty liver refers to the deposition of excess triglycerides in the liver cells. There is always a balance between
factors causing fat deposition versus factors causing fat removal from the liver

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 Factors causing fatty liver
• Mobilisation of non-esterified fatty acid(NEFA) from adipose tissue
• Reduced removal of fat from the liver
• Excess calorie intake
• Toxic injury to the liver
• Alcoholism

Why alcoholism causes fatty liver ?

In the metabolism of alcohol, it is oxidized to acetaldehyde. This reaction produces increased quantities
of NADH, which inhibits formation of oxalo-acetate from malate. As the availability of oxalo-acetate
is reduced, the oxidation Of acetyl CoA through citric acid cycle is reduced. So fatty acid accumulates
leading to TAG deposits in liver.

Past questions

✓ What are the five(5) main types of lipoproteins

✓ List the factors that tend to 1. Increase liver fat 2.Decrease liver fat
✓ Discuss the role lipoproteins in the flow of cholesterol to and fro the liver
✓ Differentiatebetween bad cholesterol and good cholesterol
✓ Write a concise note on lipid metabolism in adipose tissue
✓ Discuss cholesterol homeostasis in the cells
✓ Write a short note on reverse flow of cholesterol and its significance
✓ Write a brief note on the classification of lipoproteins

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