FOR IIT JAM 2024 ASPIRANTS

SUBJECT- BIOTECHNOLOGY

• Chapter Name- BIOCHEMISTRY

• Topic Name- Regulation of TCA cycle

Lecture No.- 20 Dr. Debasish Kar

 Topics to be covered

Topic Regulation of TCA cycle

Topic TCA cycle linked to other metabolic pathways

Topic Practice Questions

 Recap of Previous Lecture

Topic Regulation of Glycolysis

Topic: Regulation of TCA Cycle
TCA cycle – at a glance

Overall Reaction:
Acetyl-CoA + 3 NAD+ + FAD +GDP + Pi + 2 H2O
2 CO2 + 3 NADH + FADH2 + GTP + 3 H+ + CoASH

Function:
Oxidizes Acetyl-CoA into CO2 releasing
energy, which is harnessed in the
reduction of NAD+ and FAD to become
NADH and FADH2, respectively

Location:
Mitochondrial matrix (eukaryotes) or
cytoplasm (bacteria)

Rate Limiting Step:

Acetyl-CoA + OAA CoA-SH +
Citrate (Catalyzed by Citrate Synthase)
Pyruvate Dehydrogenase Complex – at a glance

Enzyme Name Cofactors Reactions Product

(*cosubstrates
)
E1 Pyruvate DH TPP 1. Decarboxylate pyruvate with TPP CO2
2.Transfer substrate to lipoamide,
which oxidizes substrate into
acetyl group and reduces
lipoamide into dihydrolipamide
E2 Dihydrolipoyl Lipoic acid, 3. CoAS - attacks acetyl substrate, Acetyl-CoA
transacetylas Coenzyme product is released
e A*
E3 Dihydrolipoyl DH FAD, NAD+* 4.Reoxidize dihydrolipoamide NADH
into lipoamide with FAD
5. Reoxidize FADH2 into FAD by NAD+
Vitamins serve as coenzymes
Structure of Acetyl CoA
Steps of TCA cycle
TCA cycle – Reaction at a glance
TCA cycle – Irreversible reactions
 TCA cycle – at a glance
TCA Cycle Matrix
SUBSTRATE PRODUCT ENZYME ENZYME REACTION TYPE
INHIBITOR
Oxaloaccetate Citrate Citrate synthase NADH, ATP, Aldol
and Acetyl CoA Citrate, Acetyl condensation
CoA
Citrate Cis-Aconitate Aconitase Fluro acetate Dehydration
Cis-Aconitate Isocitrate Aconitase Fluro acetate Hydration
Isocitrate Oxalosuccinate Isocitrate ATP Oxidation
dehydrogenase
Oxalosuccinate α-Ketoglutarate Isocitrate ATP Decarboxylation
dehydrogenase
α-Ketoglutarate Succinyl CoA α-Ketoglutarate Arsenite, NADH, Oxidative
dehydrogenase Succinyl CoA decarboxylation
Succinyl CoA Succinate Succinyl CoA Substrate level
synthetase phosphorylation
Succinate Fumarate Succinate Malonate, Oxidation
dehydrogenase Oxaloaccetate
Fumarate Malate Fumarase Hydration
Malate Oxaloaccetate Malate NADH Oxidation
dehydrogenase
 Regulation of TCA cycle
Under normal conditions, the rates of
glycolysis (PDH) and TCA cycle are
integrated so that only as much
glucose is metabolized as needed to
supply the TCA cycle with its fuel.

Basically this is an efficiency game and

you can think of ATP and NADH as
global regulators of metabolism. They
reflect metabolic flux as well as the
energy status of the cell.

Other factors in TCA cycle Regulation:

• Product Inhibition
• Substrate Availability
• Allosteric feedback inhibition of
enzyme that catalyze early steps in
cycle
 Regulation of TCA cycle

▪ Rate limiting enzyme

▪ Citrate synthase

▪ Rate controlling enzymes

▪ Citrate synthase
▪ Isocitrate dehydrogenase
▪ α‐Keoglutarate dehydrogenase

▪ Regulation by metabolic activities

▪ Substrate availability
(acetyl CoA, NAD+, FAD)
▪ Product inhibition
▪ Allosteric inhibition or activation by other
intermediates
▪ Ca+2
 Regulation of TCA cycle by Ca+2

▪ Calcium ions are the vital ions which are used during the contraction of muscle and the concomitant is
associated with the increased demand for ATP.

▪ These calcium ions can activate the enzymes like isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and
pyruvate dehydrogenase complex.

▪ Calcium ions regulate the citric acid cycle by activating pyruvate dehydrogenase, the first component of the
pyruvate dehydrogenase complex reaction that forms acetyl-CoA.
 Mechanism of regulation of TCA cycle
1) NADH/NAD+ ratio – respiratory control
The TCA cycle continues to the ETC. Reoxidation of the reduced coenzymes takes place there. In situations when is the ETC
slowed, NADH and FADH2 accumulate. It is obvious that NADH/NAD+ ratio increases and thus α-ketoglutarate
dehydrogenase and isocitrate dehydrogenase are inhibited.

2) ATP/ADP (AMP) ratio – energetic control

α-ketoglutarate dehydrogenase and isocitrate dehydrogenase are inhibited when there is a sufficient amount of energy, i.e.
ATP/ADP (AMP) ratio is high. ATP acts as inhibitor, ADP and AMP are activators of those two enzymes.

3) Availability of substrates for the TCA cycle – substrate control

As you know, velocity of the chemical reaction depends among others on the concentration of the reactants and the
products. Velocity of the TCA cycle depends on the concentration of the citrate. Activity of the citrate synthase is related to
amounts of oxaloacetate and AcCoA that are provided.

Activity of the TCA cycle is interwoven with the availability of the oxygen despite the fact that none of the reactions of the
TCA cycle require oxygen. Oxygen is vital for the ETC. Oxygen is the final acceptor of the electrons. In the ETC reoxidation
of the NADH → NAD+ and FADH2 → FAD take place. One can easily deduce that reduction of the oxygen supplies for the
cell leads to drop of concentrations of the NAD+ and FAD, hence activity of the TCA cycle is decreased.
 Amphibolic nature of TCA cycle

Catabolic role (degrading) Anabolic role (synthetic)

Acetyl CoA is
oxidized to CO2
and H2O with
releasing energy
 TCA cycle – Anaplerotic reactions

Anaplerotic reactions are chemical reactions that form intermediates of a metabolic

pathway.
 Malate Aspartate Shuttle – ATP production
▪ The malate-aspartate shuttle system, also called
the malate shuttle, is an essential system used
by mitochondria, that allows electrons to move
across the impermeable membrane between the
cytosol and the mitochondrial matrix. The
electrons are created during glycolysis, and are
needed for oxidative phosphorylation.

▪ The malate-aspartate shuttle yields

approximately 3 molecules of ATP per molecule
of cytosolic NADH and is found in liver, heart
and kidney. It is quantitatively the most
important shuttle for the reoxidation of cytosolic
NADH in vertebrate tissues under aerobic
conditions.

2.5 ATP/ NADH produced

Glycerol-3-phosphate Shuttle – ATP production

1.5 ATP/ NADH produced

▪ When cytosolic NADH transported by the glycerol 3-phosphate shuttle is oxidized by

the respiratory chain, 1.5 rather than 2.5 ATP are formed. The yield is lower because
FAD rather than NAD+ is the electron acceptor in mitochondrial glycerol 3-phosphate
dehydrogenase.
▪ The glycerol-3-phosphate shuttle allows the NADH synthesized in the cytosol by
glycolysis to contribute to the oxidative phosphorylation pathway in the mitochondria
to generate ATP. It has been found in animals, fungi, and plants.
Malate Aspartate Shuttle vs Glycerol-3-phosphate Shuttle
ATP production (complete oxidation of glucose)
TCA cycle and Glyoxylate cycle
occurs in peroxisome
TCA cycle and Urea cycle
Topic: Question analysis
 Topic: Regulation of TCA cycle

#Q. If Krebs’ cycle is overstimulated, which of the following would be overproduced?

(a) Acetyl CoA

(b) Pyruvate
(c) Glucose
(d) Carbon dioxide
 Topic: Regulation of TCA cycle
If Krebs’ cycle is overstimulated, which of the following would be
overproduced?

(a) Acetyl CoA

(b) Pyruvate
(c) Glucose
(d) Carbon dioxide

Ans. D
Of all the choices, only carbon dioxide is the product of Krebs’cycle. If the cycle is overstimulated, too
much of carbon dioxide will be produced. Glucose fuels glycolysis to produce pyruvate, which is
converted to acetyl CoA for the Krebs’ cycle. If the cycle is overstimulated, each of these would be
depleted as reactants.
 Topic: Regulation of TCA cycle
#Q. In TCA cycle malonate is competitive inhibitor structurally
similar to

(a) Succinate (b) Fumarate

(c) Oxaloacetate (d) α-keto glutarate
 Topic: Regulation of TCA cycle
In TCA cycle malonate is competitive inhibitor structurally similar to

(a) Succinate (b) Fumarate

(c) Oxaloacetate (d) α-keto glutarate

Ans. A

Malonate is a competitive inhibitor of

the enzyme succinate dehydrogenase:
malonate binds to the active site of the
enzyme without reacting, and so
competes with succinate, the usual
substrate of the enzyme.
 Topic: Regulation of TCA cycle
#Q. The energy-rich fuel molecules produced in the TCA cycle are

(a) 2 GTP, 2 NADH and 1 F ADH2

(b) 1 GTP, 2 NADH and 2 FADH2
(c) 1 GTP, 3NADH and 1 FADH2
(d) 2 GTP and 3 NADH
 Topic: Regulation of TCA cycle
The energy-rich fuel molecules produced in the TCA cycle are

(a) 2 GTP, 2 NADH and 1 F ADH2

(b) 1 GTP, 2 NADH and 2 FADH2
(c) 1 GTP, 3NADH and 1 FADH2
(d) 2 GTP and 3 NADH

Ans. C
Each acetyl coenzyme A proceeded once through the citric acid cycle. Therefore, in
total, it created 3 NADH + H+ molecules, 1 FADH2 molecules, four carbon dioxide
molecules, and 1 GTP molecule.
 Topic: Regulation of TCA cycle
#Q. Given below are statements that may or may not be correct.

A. Fructose 2, 6- biphosphate is an allosteric inhibitor of phosphofructokinase-I.

B. The TCA cycle intermediates, succinate and oxaloacetate can both be derived from
amino acids.
C. A diet rich in cysteine can compensate for a methionine deficient diet in humans.
D. dTTP for DNA synthesis can be obtained from UTP.
E. In the fatty acid biosynthetic pathway, the carbon atom from HCO3-in the synthesis of
malonyl CoA is not incorporated into palmitic acid.
Choose the option that represents the combination of all the CORRECT statements

(a) A, B , C and E (b) B, D and E

(c) A, D and E (d) Only B and C
 Topic: Regulation of TCA cycle
Given below are statements that may or may not be correct.

A. Fructose 2, 6- biphosphate is an allosteric inhibitor of phosphofructokinase-I.

B. The TCA cycle intermediates, succinate and oxaloacetate can both be derived from
amino acids.
C. A diet rich in cysteine can compensate for a methionine deficient diet in humans.
D. dTTP for DNA synthesis can be obtained from UTP.
E. In the fatty acid biosynthetic pathway, the carbon atom from HCO3-in the synthesis of
malonyl CoA is not incorporated into palmitic acid.
Choose the option that represents the combination of all the CORRECT statements

(a) A, B , C and E (b) B, D and E

(c) A, D and E (d) Only B and C

Ans. B
 Topic: Regulation of TCA cycle
#Q. Formation of glucose from acetyl CoA is called

(a) Reverse TCA cycle (b) Gluconeogenesis

(c) TCA cycle (d) Reverse Glycolysis
 Topic: Regulation of TCA cycle
Formation of glucose from acetyl CoA is called

(a) Reverse TCA cycle (b) Gluconeogenesis

(c) TCA cycle (d) Reverse Glycolysis

Ans. B

Gluconeogenesis is a metabolic pathway that results in the generation of glucose from non-
carbohydrate carbon substrates such as fatty acids (Acteyl CoA), lactate, glycerol, and glucogenic
amino acids.
 Topic: Regulation of TCA cycle
#Q. There is net gain of energy in form of ATP, if glucose which has converted into
acetyl CoA enters

(a) TCA cycle (b) Glycolysis

(c) Beta oxidation (d) Pentose phosphate pathway
 Topic: Regulation of TCA cycle
There is net gain of energy in form of ATP, if glucose which has converted into acetyl
CoA enters

(a) TCA cycle (b) Glycolysis

(c) Beta oxidation (d) Pentose phosphate pathway

Ans. A

The citric acid cycle (CAC) – also known as the TCA cycle (tricarboxylic acid cycle) or the
Krebs cycle – is a series of chemical reactions used by all aerobic organisms to release
stored energy through the oxidation of acetyl-CoA derived from carbohydrates, fats, and
proteins, into adenosine triphosphate (ATP).
 Topic: Regulation of TCA cycle
#Q. Which of the following processes occur under both aerobic and anaerobic conditions in
humans?

(a) Oxidative phosphorylation

(b) Citric acid cycle
(c) Fermentation
(d) Glycolysis
 Topic: Regulation of TCA cycle
Which of the following processes occur under both aerobic and anaerobic conditions in humans?

(a) Oxidative phosphorylation

(b) Citric acid cycle
(c) Fermentation
(d) Glycolysis

Ans. D

Glycolysis can proceed regardless of the presence of oxygen. Citric acid cycle and oxidative
phosphorylation require aerobic conditions to proceed. Fermentation only occurs under anaerobic
conditions.
 Topic: Regulation of TCA cycle
#Q. Which of the following allosteric modulators is not effective in influencing the rate of
TCA cycle ?
A) NADH
B) FADH2
C) Ca++
D) ATP
 Topic: Regulation of TCA cycle
Which of the following allosteric modulators is not effective in influencing the rate of TCA
cycle ?
A) NADH
B) FADH2
C) Ca++
D) ATP

Ans. B
 Topic: Regulation of TCA cycle
#Q. Which of the following enzyme is inhibited by Fluoroacetate in Krebs cycle ?
A) Citrate synthase
B) Aconitase
C) Isocitrate dehydrogenase
D) Fumarase
 Topic: Regulation of TCA cycle
Which of the following enzyme is inhibited by Fluoroacetate in Krebs cycle ?
A) Citrate synthase
B) Aconitase
C) Isocitrate dehydrogenase
D) Fumarase

Ans. B
 Topic: Regulation of TCA cycle
#Q. Which of the following vitamins is required for the synthesis of a co-factor required
for the conversion of succinate to fumarate ?
A) Thiamine
B) Lipoic acid
C) Pantothenic acid
D) Riboflavin
 Topic: Regulation of TCA cycle
Which of the following vitamins is required for the synthesis of a co-factor required for
the conversion of succinate to fumarate ?
A) Thiamine
B) Lipoic acid
C) Pantothenic acid
D) Riboflavin

Ans. D
2 mins Summary

1 Regulation of TCA cycle

2 TCA cycle linked to other metabolic pathways

3 Practice questions