BCH 2602 -ASS 2

QUESTION 1
1.1 Provide a comprehensive description of all the reactions that occur in the citric
acid cycle. Additionally, create a detailed graphic illustration representing the
cycle's steps.

Step 1: Oxaloacetate, a four-carbon molecule, and acetyl-CoA combine to create

citrate, a six-carbon compound. The enzyme citrate synthase is the catalyst for
this process.
Step 2: After that, by a process of hydration and hydration, citrate is isomerized
to isocitrate. This process is catalysed by the enzyme aconitase.
Step3: Alpha-ketoglutarate is created through the oxidation and decarboxylation
of isocitrate. Isocitrate dehydrogenase, an enzyme, catalyses this reaction, which
yields one molecule of carbon dioxide (CO2) and one molecule of NADH
(nicotinamide adenine dinucleotide).
Step 4: Alpha-ketoglutarate is then oxidized and decarboxylated to form succinyl-
CoA. This reaction is catalysed by the enzyme alpha-ketoglutarate
dehydrogenase complex, like the pyruvate dehydrogenase complex. One
molecule of NADH and one molecule of CO2 are produced in this step.
Step 5: Guanosine triphosphate, or GTP, is a highly energetic molecule that is
created when succinyl-CoA mixes with the molecules GDP (guanosine
diphosphate) and inorganic phosphate (Pi). The enzyme succinyl-CoA synthetase
is responsible for catalysing this reaction. Following that, the enzyme nucleoside
diphosphate kinase can convert GTP into ATP.
Step 6: CoA is taken out of succinyl-CoA to create succinate. Succinyl-CoA
synthetase is the enzyme that catalyses this reaction. In this stage, no redox
reactions take place.
Step 7: The electron transport chain enzyme succinate dehydrogenase is
responsible for oxidizing succinate to fumarate. Two hydrogen atoms are added
to the flavin adenine dinucleotide (FAD) in this process to create FADH2.
Step 8: Malate is created when the enzyme fumarase catalyses the hydration of
fumarate.
Step 9: Malate dehydrogenase converts malate to oxaloacetate, generating one
molecule of NADH in the process.
1.2 If one mole of glucose undergoes complete oxidation through glycolysis and the
citric acid cycle, calculate the total ATP yield considering substrate-level
phosphorylation and oxidative phosphorylation.

1. Glycolysis: The initial stage of the metabolism of glucose is glycolysis. Through

the process of glycolysis, one mole of glucose creates two moles of pyruvate.
Two moles of ATP are produced during glycolysis by substrate-level
phosphorylation.

Glycolysis generates 2 moles of ATP net.

2.Each mole of pyruvate enters the citric acid cycle, sometimes referred to as the
Krebs cycle, through the process of oxidation. Pyruvate is transformed into
Acetyl-CoA before to entering the cycle, producing one mole of NADH. We will
have two moles of NADH because one mole of glucose produces two moles of
pyruvate.

No ATP is produced as a result of pyruvate oxidation.

3. Citric Acid Cycle: Three molecules of NADH, one molecule of FADH2, and one
molecule of GTP (which can be transformed directly into ATP) are created for
every Acetyl-CoA molecule that enters the citric acid cycle.

10 moles of ATP are produced by the citric acid cycle overall (3 NADH x 2.5 ATP,
1 FADH2 x 1.5 ATP, and 1 GTP x 1 ATP).

4. Oxidative Phosphorylation: The electron transport chain (ETC) in the

mitochondria is where the NADH and FADH2 produced during glycolysis,
pyruvate oxidation, and the citric acid cycle enter. The ETC uses oxidative
phosphorylation to produce ATP.

Each NADH produces around 2.5 ATP, whereas each FADH2 produces about 1.5
ATP.

The amount of NADH and FADH2 generated by the metabolism of glucose

determines the net ATP produced by oxidative phosphorylation.

Each NADH is thought to produce about 2.5 ATP under normal circumstances,
while each FADH2 is thought to produce about 1.5 ATP. Therefore, we can
calculate the ATP production from oxidative phosphorylation to be roughly 2.5
ATP per NADH and 1.5 ATP per FADH2 if we assume equal levels of both NADH
and FADH2.
 As a result, the ATP yield from oxidative phosphorylation would be as follows if
we assumed two moles of NADH and two moles of FADH2 (since we had two
moles of each from glucose metabolism):

Net ATP produced by oxidative phosphorylation is equal to 10 ATP (2 NADH x 2.5

ATP + 2 FADH2 x 1.5 ATP).
ATP yields from each process added together:

Net ATP from glycolysis plus Net ATP from pyruvate oxidation, Net ATP from the
citric acid cycle, and Net ATP from oxidative phosphorylation make up the total
amount of ATP produced.
Total ATP produced: 22 ATP from 2 ATP, 0 ATP, 10 ATP, and 10 ATP.

Therefore, considering substrate-level phosphorylation and oxidative

phosphorylation, the total ATP production from the full oxidation of one mole of
glucose through glycolysis and the citric acid cycle is 22 ATP.

1.3 Identify the regulatory mechanisms that control the citric acid cycle. Analyse how
these mechanisms impact the cycle's activity and metabolic pathway.

• Availability of substrates: The citric acid cycle needs acetyl-CoA and

oxaloacetate as substrates in order to function. The cycle is greatly
influenced by how readily available these substrates are. Numerous
processes, such as the metabolism of glucose, fatty acids, and amino acids,
produce acetyl-CoA. Other metabolic processes like glycolysis and fatty acid
oxidation control the levels of acetyl-CoA and oxaloacetate. As a result, the
availability of substrates for the citric acid cycle may be affected by the pace
of these routes.
• Feedback inhibition: To control their own production, a number of
intermediate molecules in the citric acid cycle function as allosteric inhibitors.
For instance, the buildup of ATP and NADH, which are generated during the
cycle, might prevent important cycle enzymes like isocitrate dehydrogenase
and alpha-ketoglutarate dehydrogenase from working. By avoiding excessive
ATP and NADH generation, this feedback inhibition aids in maintaining the
balance of energy production.
• Enzyme regulation: Covalent modifications, such as phosphorylation or
dephosphorylation, can be used to control the activity of enzymes involved in
the citric acid cycle. Protein kinases are responsible for the phosphorylation
of enzymes, which can either activate or inhibit an enzyme's function. For
instance, pyruvate dehydrogenase kinase can block pyruvate
dehydrogenase, which converts pyruvate to acetyl-CoA before entering the
cycle. This control enables the citric acid cycle's activity to be tailored based
on the cell's energy requirements.
• Hormonal control: Hormones like glucagon and insulin can affect how active
the citric acid cycle is. High blood glucose levels cause the release of insulin,
which activates cycle enzymes to promote glucose oxidation and ATP
synthesis. In contrast, glucagon, which is released when blood sugar levels
are low, blocks the citric acid cycle to save glucose and encourage the use of
alternate fuels like fatty acid oxidation. This hormonal control aids in adjusting
the cycle's activity to the organism's general metabolic status.
1.4 Provide a detailed explanation of the glyoxylate pathway's reactions.
Additionally, create a graphical representation of the pathway's steps.
Step 1: Just like in the citric acid cycle, acetyl-CoA often condenses with
oxaloacetate to create citrate in the glyoxylate cycle.
Step 2: In the second stage, citrate releases one water molecule and
transforms into cis-aconitate. An enzyme called aconitase, which is involved
in the citric acid cycle, catalyses the reaction.
Step 3: Isocitrate is created by catalytic hydrolysis of cis-aconitate. The
aconitase enzyme catalyses this process.
Step 4: In this process, isocitrate is not broken down through isocitrate
dehydrogenation, but rather through a cleavage reaction that creates
succinate and glyoxylate. The isocitrate lyase enzyme is responsible for
catalysing the reaction.
Step 5: As a result, the enzyme malate synthetase catalyses the reaction in
which the produced glyoxylate condenses with acetyl-CoA to produce malate.
Step 6: the malate is further oxidized to produce oxaloacetate, which can
then combine with an additional acetyl-CoA molecule to complete the cycle.
 1.5. Describe the anapleurotic reactions and provide molecular structures for
the key compounds involved.
1. Pyruvate to Oxaloacetate Conversion: Catalysed by the enzyme pyruvate
carboxylase, this reaction necessitates the cofactor biotin. Using one molecule of
ATP, pyruvate carboxylase carboxylates pyruvate to create oxaloacetate. Pyruvate's
molecular structure is as follows:

And the molecular structure of oxaloacetate is:

2. Propionyl-CoA to Succinyl-CoA Conversion:
Propionyl-CoA carboxylase, an enzyme, and the cofactor biotin are both
necessary for this reaction to occur. The carboxylation of propionyl-CoA results in
the formation of D-methyl malonyl-CoA, which is isomerized to L-methyl malonyl-
CoA. The enzyme methyl malonyl-CoA mutase, which needs the cofactor vitamin
B12, then transforms L-methyl malonyl-CoA into succinyl-CoA. Propionyl-CoA is
made up of the following molecules:

And the molecular structure of succinyl-CoA is:

QUESTION 2

1.1. Explain the reactions involved in gluconeogenesis and compare them to

those of glycolysis.
When glucose levels are low, gluconeogenesis produces glucose from sources
other than carbohydrates. Glycolysis breaks down glucose to produce energy.

In the cytoplasm of cells, a sequence of events known as glycolysis take place. It

entails the conversion of two molecules of pyruvate, a three-carbon molecule,
from the six-carbon molecule glucose. There are ten steps in this process, and
unique enzymes catalyse each one of them. A tiny amount of ATP (adenosine
triphosphate) and NADH (nicotinamide adenine dinucleotide) are produced
throughout the process of glycolysis.

The production of glucose from non-carbohydrate precursors such lactate,

pyruvate, amino acids, and glycerol is known as gluconeogenesis, on the other
hand. The liver and kidneys play a smaller role in glucose synthesis than the liver.
It is essentially the opposite of glycolysis, but with a few additional enzymes and
reactions to get around glycolysis's irreversible processes.

While glycolysis occurs in the cytoplasm, some gluconeogenesis reactions also

take place there, while others take place in the mitochondria. The three
irreversible steps of glycolysis, which are performed by the enzymes hexokinase,
phosphofructokinase, and pyruvate kinase, are skipped during the processes of
gluconeogenesis. In the process of gluconeogenesis, various enzymes and
processes take their place.

Although the processes involved in glycolysis and gluconeogenesis share some

steps, they differ in certain enzymes and reactions to ensure that glycolysis is
reversed, and glucose is produced instead of broken down.

1.2 Describe the mechanisms employed in the regulation of gluconeogenesis.

• Hormonal Regulation: Hormones have a big impact on how

gluconeogenesis is controlled. Glucagon and insulin are two important
hormones in play. In reaction to low blood glucose levels, the pancreas
releases glucagon, which increases gluconeogenesis in the liver. Inhibiting
gluconeogenesis and promoting glucose storage are the effects of insulin,
which is released in response to elevated blood sugar levels.
• Availability of Substrates: The availability of the substrates needed for
gluconeogenesis has an impact on how it is regulated. Several precursors
are transformed into glucose, including lactate, pyruvate, glycerol, and
certain amino acids. The amount of these substrates in the body and how
quickly they enter the liver have an impact on how quickly
gluconeogenesis occurs.
• Enzyme Regulation: Several strategies are used to control the enzymes
involved in gluconeogenesis. Fructose-1,6-bisphosphatase (FBPase), for
instance, is a crucial regulatory enzyme that prevents gluconeogenesis.
Fructose-2,6-bisphosphate, which is managed by the enzyme
phosphofructokinase-2 (PFK-2), controls its activity. By modulating the
activity of PFK-2, hormones like glucagon and insulin indirectly control the
activity of FBPase and gluconeogenesis.
• Transcriptional Control: At the transcriptional level, the expression of
genes coding for gluconeogenic enzymes is controlled. The transcription
of crucial gluconeogenic enzymes is activated by transcription factors
including FOXO1 (Forkhead box protein O1) and CREB (cAMP response
element-binding protein). These transcription factors can be activated or
inhibited by hormones and signaling pathways, which affects
gluconeogenesis.
• Energy condition: Gluconeogenesis is also influenced by the energy
condition of the cell. An energy imbalance is indicated by low levels of ATP
or high levels of AMP or ADP, which triggers gluconeogenesis to produce
glucose for energy.
1.3. Represent all the reactions, with enzymes and coenzymes, involved in
the biosynthesis of glycogen by means of balanced chemical equations.
1. Glucose-6-Phosphate Conversion:
Glucose-6-phosphate (G6P) is converted to glucose-1-phosphate (G1P) by
the enzyme phosphoglucomutase (PGM) with the help of the coenzyme
glucose-1,6-bisphosphate.

G6P ⇌ G1P

Enzyme: Phosphoglucomutase (PGM)

Coenzyme: Glucose-1,6-bisphosphate
2. Activation of Glucose-1-Phosphate:
G1P is activated by the enzyme UDP-glucose pyrophosphorylase (UGPase)
with the help of the coenzyme UTP, resulting in the formation of UDP-glucose.
G1P + UTP ⇌ UDP-glucose + PPi

Enzyme: UDP-glucose pyrophosphorylase (UGPase)

Coenzyme: UTP
3. Glycogen Chain Elongation:
Glycogen synthase catalyses the transfer of glucose from UDP-glucose to the
growing glycogen chain, forming a new α-1,4-glycosidic bond.

UDP-glucose + (glycogen)n ⇌ UDP + (glycogen)n+1

Enzyme: Glycogen synthase

Coenzyme: None required
4. Branching of Glycogen Chains:
Glycogen branching enzyme catalyses the transfer of a segment of the
glycogen chain to form a new α-1,6-glycosidic bond, creating branching points
in the glycogen molecule.
(glycogen)n ⇌ (glycogen)n+1

Enzyme: Glycogen branching enzyme

Coenzyme: None required
1.4. Identify and describe the mechanisms used to control the rate of synthesis of
glycogen.

• Hormonal control: The regulation of glycogen production depends heavily on

hormones like insulin and glucagon. By activating crucial enzymes involved in
glycogen production and inhibiting enzymes involved in glycogen breakdown,
insulin, which is released in response to high blood glucose levels, promotes
glycogen synthesis. On the other hand, glucagon, which is released when
blood sugar levels are low, encourages the breakdown of glycogen to release
glucose while inhibiting its production.
• Allosteric regulation: Allosteric regulation is the process by which certain
chemicals attach to enzymes and change the activity of the enzymes.
Allosteric control of glycogen production occurs when substances like
glucose-6-phosphate (G6P) and ATP bind to important enzymes like glycogen
synthase and glycogen phosphorylase. Low concentrations of G6P and ATP
activate glycogen synthase, increasing glycogen synthesis, whereas high
concentrations of G6P and ATP block glycogen synthase, preventing further
glycogen synthesis.
• Covalent modification: Enzymes involved in the process are covalently
modified to control glycogen production. The major enzymes involved in the
manufacture and breakdown of glycogen, glycogen synthase and glycogen
phosphorylase, are controlled by the processes of phosphorylation and
dephosphorylation. Glycogen synthase's activity is inhibited by
phosphorylation while it is activated by dephosphorylation. On the other hand,
dephosphorylation inhibits glycogen phosphorylase, whereas phosphorylation
promotes glycogen breakdown.
• Accessibility of substrates: The rate of glycogen synthesis is also influenced
by the accessibility of substrates like glucose-6-phosphate and UDP-glucose.
As these molecules are used as the building blocks for glycogen molecules,
high concentrations of glucose-6-phosphate and UDP-glucose encourage
glycogen production. On the other hand, a lack of these substrates can
restrict the production of glycogen.
1.5 Explain the physiological need for glucose synthesis in animals.
• Energy production: Glucose is an essential energy source for many tissues,
particularly the brain, which depends on it substantially. Glucose is converted
into ATP, the universal energy unit of cells, through the processes of glycolysis
and the citric acid cycle. whether dietary glucose is scarce, whether fasting for
an extended period or when exercising, glucose synthesis is essential to
maintain a steady supply of energy.
• Blood glucose level maintenance: To guarantee a steady supply of glucose to
tissues that depend on it, blood glucose levels must be strictly controlled.
Gluconeogenesis, another name for the process of making glucose, is
predominantly carried out by the liver and, to a lesser extent, by the kidneys.
Gluconeogenesis aids in keeping blood glucose levels within a normal range
when fasting, when glucose from the food is not available. This is essential to
avoid hypoglycaemia, which can cause other metabolic problems like reduced
brain function.
• Refuelling with glycogen: The liver and muscles produce and store glycogen,
a storage form of glucose. Extra glucose is turned into glycogen for storage
during times of high glucose availability. Glycogen is broken down through a
process called glycogenolysis to release glucose and maintain blood glucose
levels when levels fall. After glycogenolysis has taken place, glucose
synthesis is necessary to replace glycogen stores and maintain a steady
supply of glucose for energy production.
• Red blood cells (RBCs) need glucose as their only source of energy because
they lack mitochondria. RBCs use the process of glycolysis to break down
glucose into ATP, which is necessary for the RBCs' job of carrying oxygen
throughout the body. Glucose synthesis assists
in supplying RBCs with glucose, maintaining their correct operation.
1.6 Describe the stoichiometry and energy balance in gluconeogenesis.
• Stoichiometry:
1. Pyruvate carboxylase reaction: Pyruvate + CO2 + ATP → Oxaloacetate +
ADP + Pi
2. Phosphoenolpyruvate carboxykinase reaction: Oxaloacetate + GTP →
Phosphoenolpyruvate + GDP + CO2
3. Fructose-1,6-bisphosphatase reaction: Fructose 1,6-bisphosphate + H2O
→ Fructose 6-phosphate + Pi
4. Glucose-6-phosphatase reaction: Glucose 6-phosphate + H2O → Glucose
+ Pi
Through the process of gluconeogenesis, two molecules of pyruvate are
ultimately changed into one molecule of glucose. Other non-carbohydrate
precursors can be utilized in addition to pyruvate, and their precise
stoichiometry may differ.
• Energy Balance: Gluconeogenesis requires the input of both ATP and GTP,
making it an energy intensive process. The following is a succinct summary of
the net energy balance:

One ATP molecule is needed for the pyruvate carboxylase process, and two
ATP molecules are utilized for the phosphoenolpyruvate carboxykinase
reaction, totalling three ATP molecules used during gluconeogenesis.

2. GTP consumption: The phosphoenolpyruvate carboxykinase process uses

up one molecule of GTP.

3. creation of NADH and FADH2: The conversion of 2 molecules of lactate to

pyruvate results in the creation of 2 molecules of NADH, and the conversion
of 2 molecules each of oxaloacetate to malate and malate to oxaloacetate
results in the production of 2 molecules each of NADH and FADH2. The
electron transport chain can then be joined by these NADH and FADH2
molecules, aiding in the production of ATP.

Gluconeogenesis's overall energy balance can be summed up as being ATP-

and GTP-consuming, but it also produces NADH and FADH2, which can help
produce ATP through oxidative phosphorylation. The energy balance ensures
that the gluconeogenesis process is energetically advantageous and can take
place even when there is a shortage of glucose and energy demands are
high.
1.7 Explain the roles of extrahepatic phosphoenolpyruvate carboxykinase (PEPCK)
in gluconeogenesis.
Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme that catalyses a crucial
step in the production of glucose from non-carbohydrate sources, or
gluconeogenesis.
PEPCK exists in two different isoforms: cytosolic PEPCK1 and mitochondrial
PEPCK2. While PEPCK2 is expressed throughout the body, PEPCK1 is mostly
found in the liver and kidney.
Oxaloacetate is transformed by PEPCK1 into phosphoenolpyruvate and carbon
dioxide with GTP acting as a cofactor.
Oxaloacetate is transformed by PEPCK2 into phosphoenolpyruvate and carbon
dioxide with ATP serving as a cofactor.
Hormones, transcription factors, and metabolites control PEPCK to keep blood sugar
levels normal.
1.8 Discuss the reciprocal regulation of glycolysis and gluconeogenesis.
The same substance or treatment might have opposing effects on the two metabolic
pathways of glycolysis and gluconeogenesis since these two metabolic processes
are reciprocally regulated.
For instance, although ATP and citrate increase glycolysis and inhibit
gluconeogenesis, AMP and fructose-2,6-bisphosphate (F2,6BP) have the opposite
effects.
This stops a pointless cycle of simultaneously generating and destroying glucose.
1.9. Explain the role of fructose-2,6-bisphosphate in the control of gluconeogenesis.
The metabolite fructose-2,6-bisphosphate (Fru-2,6-P 2) influences the activity of the
enzymes fructose 1,6-bisphosphatase (FBPase-1) and phosphofructokinase 1 (PFK-
1) to control glycolysis and gluconeogenesis. Fru-2,6-P 2 increases glycolysis and
decreases gluconeogenesis via stimulating PFK-1 and inhibiting FBPase-1. Both
blood glucose levels and hormonal state affect the concentration of Fru-2,6-P 2.
1.10 Describe the roles of glycogen synthase and the branching process in glycogen
biosynthesis.
Glycogen biosynthesis depends heavily on glycogen synthase and glycogen
branching enzyme. The chain is extended by glycogen synthase using 1,4-glycosidic
bonds to add glucose. The creation of branches by the glycogen branching enzyme
results in a more compact macromolecule and more effective energy storage.