The Krebs cycle, also known as the citric acid cycle or TCA cycle, is a series of chemical reactions in the mitochondria that break down food for energy. It is the final common pathway that produces ATP through oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. The cycle generates high-energy electrons in the form of NADH and FADH2 that are used to produce ATP through oxidative phosphorylation. Hyperammonemia can lead to loss of consciousness by withdrawing alpha-ketoglutarate from the TCA cycle to form glutamine, lowering ATP production.

1. Krebs Cycle
Dr. Roshan Kumar Mahat, PhD

2. At the end of the class, students should be
able to:
1. Describe the reactions of TCA cycle and the
reactions that lead to the production of
reducing equivalents that are oxidized in the
mitochondrial electron transport chain to
yield ATP.
2. Describe the regulations of TCA cycle.
3. Explain the importance of vitamins in the
citric acid cycle.

3. 4. Explain how the citric acid cycle provides
both a route for catabolism of amino acids
and also a route for their synthesis.
5. Describe the anaplerotic reactions of TCA
cycle.
6. Describe the role of TCA cycle in fatty acid
synthesis.
7. Describe the inhibitors of TCA cycle.
8. Explain how hyperammonemia can lead to
loss of consciousness.

4. Also called TCA cycle or citric acid cycle.
The citric acid cycle is a sequence of reactions
in mitochondria that result in the oxidation of
an acetyl group to two molecules of carbon
dioxide and reduces the coenzymes that are
reoxidized through the electron transport
chain, linked to the formation of ATP.
Final common oxidative pathway for the
oxidation of carbohydrate, lipid and protein.
Introduction

5. Overview of TCA cycle

6. Role of oxaloacetate in citric acid cycle
The four-carbon molecule, oxaloacetate that
initiates the first step in the citric acid cycle is
regenerated at the end of one passage through
the cycle.
The oxaloacetate acts catalytically: it
participates in the oxidation of the acetyl group
but is itself regenerated.
Thus, only a small quantity of oxaloacetate is
needed for the oxidation of a large quantity of
acetyl CoA molecules.

7. Location of enzymes of TCA cycle
The enzymes of the TCA cycle are located in
the mitochondrial matrix except succinate
dehydrogenase which is located in the inner
mitochondrial membrane.
Steps/reactions of TCA cycle
TCA cycle consists of eight sequential
reactions.

8. Step 1: Formation of citrate

9. Step 2: Isomerization of citrate

10. Step 3: Oxidative decarboxylation of
isocitrate

11. Step 4: Oxidative decarboxylation of
alpha-ketoglutarate

12. Step 5: Cleavage of succinyl CoA

13. Step 6: Oxidation of succinate

14. Step 7: Hydration of fumarate

15. Step 8: Oxidation of malate

17. Energetics of TCA cycle
As a result of oxidations catalyzed by the
dehydrogenases of the citric acid cycle, three
molecules of NADH and one of FADH2 are
produced for each molecule of acetyl-CoA
catabolized in one turn of the cycle.
These reducing equivalents are transferred to the
respiratory chain, where reoxidation of each
NADH results in formation of 2.5 ATP, and of
FADH2, 1.5 ATP.
In addition, 1 ATP (or GTP) is formed by
substrate-level phosphorylation catalyzed by
succinate thiokinase.

18. ATP Generation Steps of Citric Acid Cycle
Step No. Reactions no. Co-enzyme ATPs gene-
rated
3 Isocitrate → alpha ketoglutarate NADH 2.5
4 Alpha ketoglutarate → succinyl CoA NADH 2.5
5 Succinyl CoA → Succinate GTP 1
6 Succinate → Fumarate FADH2 1.5
8 Malate → Oxaloacetate NADH 2.5
Total 10

19. Steps where energy is trapped are marked with the
coenzyme and the number of ATP generated during that
reaction. A total of 10 ATPs are generated during one cycle.

20. Total ATP formation in the
catabolism of glucose

21. Vitamins play key roles in the citric acid cycle
Four of the B vitamins are essential in the citric acid cycle
and hence energy-yielding metabolism:
1. Riboflavin, in the form of flavin adenine dinucleotide
(FAD), a cofactor for succinate dehydrogenase;
2. Niacin, in the form of nicotinamide adenine
dinucleotide (NAD+), the electron acceptor for
isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, and malate dehydrogenase;
3. Thiamin (vitamin B1), as thiamin diphosphate, the
coenzyme for decarboxylation in the α-ketoglutarate
dehydrogenase reaction; and
4. Pantothenic acid, as part of coenzyme A, such as
Acetyl CoA and Succinyl CoA.

22. Regulation of the TCA cycle
1. REGULATION OF PDH COMPLEX
2. REGULATION OF TCA CYCLE ENZYMES
1. Citrate synthase
2. Isocitrate dehydrogenase
3. α-ketoglutarate dehydrogenase
4. Succinate dehydrogenase

23. Regulation of PDH complex
Activity of pyruvate dehydrogenase complex
is switched on or switched off based on the
cellular energy needs.
Two mechanisms of regulations have been
recognised.
1. Allosteric regulation
2. Covalent modulation

24. Allosteric regulation
PDH complex is inhibited
by acetyl-CoA and NADH
and activated by non-
acetylated CoA (CoASH)
and NAD+.

25. Covalent modification
The pyruvate dehydrogenase
activities of the PDH complex are
regulated by their state of
phosphorylation. This modification is
carried out by a specific kinase (PDH
kinase) and the phosphates are
removed by a specific phosphatase
(PDH phosphatase).
PDH kinase is activated by NADH
and acetyl-CoA and inhibited by
pyruvate, ADP, CoASH, Ca2+ and
Mg2+.
The PDH phosphatase, in contrast, is
activated by Mg2+ and Ca2+.

26. Regulation of TCA cycle enzymes
The most likely sites for regulations are the
nonequilibrium reactions catalyzed citrate
synthase, isocitrate dehydrogenase, and α-
ketoglutarate dehydrogenase.
The dehydrogenases are activated by Ca2+,
which increases in concentration during
contraction of muscle and during secretion by
other tissues, when there is increased energy
demand.

27. A. Citrate synthase-
There is allosteric inhibition of citrate synthase
by succinyl CoA, NADH, ATP and long-chain
fatty acyl-CoA.
B. Isocitrate dehydrogenase-
is allosterically stimulated by ADP, which
enhances the enzyme's affinity for substrates.
In contrast, NADH and ATP inhibits iso-citrate
dehydrogenase.

28. C. α-ketoglutarate dehydrogenase –
α- Ketoglutarate dehydrogenase is inhibited by
succinyl CoA and NADH. In addition, α-
ketoglutarate dehydrogenase is inhibited by a
high energy charge. Thus, the rate of the cycle is
reduced when the cell has a high level of ATP.
D. Succinate dehydrogenase-
is inhibited by oxaloacetate, and the availability
of oxaloacetate, as controlled by malate
dehydrogenase, depends on the
[NADH]/[NAD+] ratio.

29. Regulation of TCA cycle (summary)
molecules of higher
energy state i.e. ATP,
NADH, citrate, Acetyl
CoA ----------------------
inhibit TCA cycle
molecules of low
energy state i.e. ADP,
AMP, NAD+-----------
stimulate TCA cycle

30. Significance of Citric Acid Cycle
1. Complete oxidation of acetyl-CoA
2. ATP generation
3. Final common oxidative pathway
4. Integration of major metabolic pathways
5. Fat is burned on the wick of carbohydrates
6. Excess carbohydrates are converted as neutral fat
7. No net synthesis of carbohydrates from fat
8. Carbon skeletons of amino acids finally enter the citric acid
cycle
9. Amphibolic pathway
10. Anaplerotic role

31. 1. Complete oxidation of acetyl CoA:
2. ATP generation:
Each cycle of TCA produces 10 molecules of
ATP.

32. 3. Final common oxidative pathway

33. 4. Integration of major metabolic pathways

34. 5. Fats burn in the flame of carbohydrates
Fats burn in the flame of carbohydrates means
fats can only be oxidized in the presence of
carbohydrates.
Acetyl CoA represents fat component, since
the major source is fatty acid oxidation.
Acetyl CoA is completely oxidized in the TCA
cycle in the presence of oxaloacetate.

35. Pyruvate is mainly used up for Anaplerotic
reactions to compensate for oxaloacetate
concentration.
Thus without carbohydrates (Pyruvate), there
would be no Anaplerotic reactions to replenish
the TCA cycle components.
With a diet of fats only, the acetyl CoA from fatty
acid degradation would not get oxidized and
build up due to non functioning of TCA cycle.
Thus fats can burn only in the flame of
carbohydrates.

36. 6. Excess carbohydrates are converted as
neutral fat
The pathway is glucose to pyruvate to acetyl
CoA to fatty acid. However, fat can not be
converted to glucose because pyruvate
dehydrogenase reaction is an absolutely
irreversible step.
Glucose  Pyruvate  Acetyl Co A
Fatty acid

37. 7. No net synthesis of carbohydrates from fat
Acetyl CoA entering in the cycle is completely
oxidized to CO2 by the time the cycle reaches
Succinyl CoA. So acetyl CoA can not be used
for the synthesis of carbohydrates. Thus, acetyl
CoA can not be used for gluconeogenesis.
Fatty acid  Acetyl Co A  CO2
Glucose

38. 8. Amino acids finally enters the TCA cycle

39. 9. TCA cycle: an amphibolic pathway
The citric acid cycle is not only a pathway for
oxidation of two-carbon units, but is also a major
pathway for interconversion of metabolites arising
from transamination and deamination of amino acids,
and providing the substrates for amino acid synthesis
by transamination, as well as for gluconeogenesis and
fatty acid synthesis.
Because it functions in both oxidative and synthetic
processes, it is amphibolic.

40. I. Catabolic role of TCA cycle
The citric acid cycle is the final common pathway
for the oxidation of carbohydrate, lipid, and
protein because glucose, fatty acids, and most
amino acids are metabolized to acetyl-CoA or
intermediates of the cycle.
The function of the citric acid cycle is the
harvesting of high-energy electrons from carbon
fuels.
1 acetyl CoA molecule generates approximately
10 molecules of ATP per turn of the cycle.

41. II. Anabolic role of TCA cycle
a) Glucose biosynthesis
Gluconeogenesis, which occurs in the
cytosol, utilizes oxaloacetate as its starting
material. Oxaloacetate is not transported
across the mitochondrial membrane, but
malate is.
Malate that has been transported across the
mitochondrial membrane is converted to
oxaloacetate in the cytosol for
gluconeogenesis.

42. b) Amino acid biosynthesis
utilizes citric acid cycle intermediates in two ways. -
Ketoglutarate is converted to glutamate in a reductive
amination reaction involving either NAD or NADP
catalyzed by glutamate dehydrogenase.
Alpha Ketoglutarate and oxaloacetate are also used to
synthesize glutamate and aspartate in transamination
reactions.

43. c) Fatty acid synthesis
Acetyl-CoA, formed from pyruvate by
the action of pyruvate dehydrogenase, is
the major substrate for long-chain fatty
acid synthesis.
Acetyl CoA can also be used for the
synthesis of cholesterol, steroids etc.

44. d) Heme synthesis
Succinyl CoA
condenses with
amino acid
Glycine to form
Alpha amino beta
keto Adipic acid,
which is the first
step of heme
biosynthesis.

45. e) Purine and pyrimidine synthesis
Glutamate and Aspartate derived from TCA cycle
are utilized for the synthesis of purines and
pyrimidines.

46. Summary of anabolic role of TCA cycle

47. 10.Anaplerotic role of TCA cycle
“Filling up” reactions or “influx” reactions or
“replenishing” reactions
Anaplerosis is the act of replenishing TCA
cycle intermediates that have been extracted
for biosynthesis (in what are called
cataplerotic reactions).
Anaplerotic flux must balance cataplerotic flux
in order to retain homeostasis of cellular
metabolism

48. 1. Formation of oxaloacetate from pyruvate
Pyruvate can be converted to oxaloacetate by
pyruvate carboxylase.
2. Formation of malate from pyruvate
Pruvate can be converted to malate by
NADP+ dependent malic enzyme.
3. Formation of oxaloacetate from aspartate
Oxaloacetate can also be formed from
aspartate by transamination reaction.

49. 4. Formation of Alpha keto glutarate
Alpha ketoglutarate can be formed from
Glutamate by glutamate dehydrogenase or by
transamination reactions.
5. Formation of fumarate
Fumarate can be formed from phenylalanine
and tyrosine.

50. 5. Formation of Succinyl CoA
Succinyl CoA can be produced from the
oxidation of odd chain fatty acid and from the
metabolism of valine, methionine and
isoleucine (through carboxylation of Propionyl
CoA to Methyl malonyl CoA and then
Succinyl CoA)

52. Inhibitors of TCA cycle
Inhibitor Enzyme inhibited Mechanism
Fluoroacetate Aconitase Suicide inhibition
Arsenite Alpha ketoglutarate
dehydrogenase
Non-competitive
inhibition
Malonate Succinate
dehydrogenase
Competitive
inhibition

53. Some Mutations in Enzymes of the Citric Acid
Cycle Lead to Cancer
Mutations in citric acid cycle enzymes are very
rare in humans and other mammals, but those
that do occur are devastating. Genetic defects
in the fumarase gene lead to tumors of smooth
muscle (leiomas) and kidney; mutations in
succinate dehydrogenase lead to tumors of the
adrenal gland (pheochromocytomas).

54. Another remarkable connection between citric
acid cycle intermediates and cancer is the
finding that in many glial cell tumors
(gliomas), the NADPH dependent isocitrate
dehydrogenase has an unusual genetic defect.
The inhibition of the histone demethylases
in turn interferes with normal gene
regulation, leading to unrestricted glial cell
growth
Alpha ketoglutarate and Fe3+ are essential
cofactors for a family of histone
demethylases that alter gene expression

55. Hyperammonemia, as occurs in advanced liver disease
leads to loss of consciousness, coma, convulsions and
may be fatal --Justify
This is largely because of the withdrawal of alpha-
ketoglutarate to form glutamate (catalyzed by
glutamate dehydrogenase) and then glutamine
(catalyzed by glutamine synthetase), leading to
lowered concentrations of all citric acid cycle
intermediates, and hence reduced generation of ATP.
In addition ammonia inhibits alpha ketoglutarate
dehydrogenase, and possibly also pyruvate
dehydrogenase.