The citric acid cycle (TCA cycle) is a central metabolic pathway that oxidizes acetyl-CoA derived from carbohydrates, fats, and proteins, producing carbon dioxide and reducing equivalents in the form of NADH and FADH2. The TCA cycle consists of 8 steps that occur in the mitochondrial matrix and ultimately generate 12 ATP per acetyl-CoA molecule. Regulation of the TCA cycle occurs through three enzymes and is influenced by levels of ATP, NADH, and other metabolites. While the TCA cycle functions primarily in energy production, it also interfaces with many anabolic pathways through various intermediates.

1. TCA CYCLE STEPS
REGULATION AND
SIGNIFICANCE
PRESENTED BY
B.RASHMI
M.Sc. BIOCHEMISTRY

2. Introduction
• The citric acid cycle is the central metabolic
hub of the cell.
• It is the final common pathway for the
oxidation of fuel molecule such as amino
acids, fatty acids, and carbohydrates.
• In eukaryotes, the reactions of the citric acid
cycle take place inside mitochondria, in
contrast with those of glycolysis, which take
place in the cytosol.

3. Overview of the Citric Acid Cycle
• The citric acid cycle (Krebs cycle,
tricarboxylic acid cycle) includes a series of
oxidation-reduction reactions in mitochondria
that result in the oxidation of an acetyl group
to 2 molecules of CO2 and reduce the
coenzymes that are reoxidized through the
ETC, linked to the formation of ATP.

4. • A 4C compound (OAA) condenses with 2C acetyl
unit to yield a 6C tricarboxylic acid (citrate).
• An isomer of citrate is then oxidatively
decarboxylated.
• The resulting 5C compound (α-ketoglutarate) also
is oxidatively decarboxylated to yield a 4C
compound (succinate).
• OAA is then regenerated from succinate.
• 2C atoms enter the cycle as an acetyl unit and two
carbon atoms leave the cycle in the form of two
molecules of CO2.

5. • Three hydride ions
(hence, 6 electrons) are
transferred to 3 molecules
of NAD+, whereas 1pair
of hydrogen atoms
(hence, 2 electrons) are
transferred to one
molecule of FAD.
• The function of the citric
acid cycle is the
harvesting of high energy
electrons from carbon
fuels.

6. Requirement of O2 by TCA cycle
• Oxygen is required for the citric acid cycle
indirectly in as much as it is the electron
acceptor at the end of the ETC, necessary to
regenerate NAD+ and FAD.

7. Requirement of O2 by TCA cycle
• There is no direct participation of O2 in TCA
cycle.
• Operates only under aerobic conditions.
• Therefore, citric acid cycle is strictly aerobic

8. Role of oxaloacetate in citric
acid cycle
• The 4C molecule, OAA that initiates the 1st step
in the citric acid cycle is regenerated at the end
of one passage through the cycle.
• The OAA acts catalytically it participates in
the oxidation of the acetyl group but is itself
regenerated.
• Thus, 1 molecule of OAA is capable of
participating in the oxidation of many acetyl
molecules.

9. TCA CYCLE

10. Reactions of the Citric Acid
Cycle
Step-1 Formation of Citrate-
(Condensation)
• The citric acid cycle begins with the
condensation of a 4C unit, OAA, and a 4C
unit, the acetyl group of acetyl CoA. OAA
reacts with acetyl CoA and H2O to yield
citrate and CoA.
• This reaction, which is an aldol condensation
followed by a hydrolysis, is catalyzed by
citrate synthase.

11. OAA first condenses with acetyl CoA to
form citryl CoA, which is then hydrolyzed
to citrate and CoA

12. Step-2-Formation of Isocitrate
(Isomerization)
• Citrate is isomerized into isocitrate to enable the
6C unit to undergo oxidative decarboxylation.
• The isomerization of citrate is accomplished by a
dehydration step followed by a hydration step.
• The result is an interchange of a hydrogen atom
and a hydroxyl group.
• The enzyme catalyzing both steps is called
Aconitase because cis-aconitate is an intermediate.

13. Aconitase is an iron-sulfur protein, or nonheme iron
protein. It contains 4 iron atoms that are not
incorporated as part of a heme group.

14. Step-3- Formation of α- Keto
Glutarate (Oxidative
decarboxylation)
• Isocitrate undergoes dehydrogenation catalyzed by
isocitrate dehydrogenase to form, initially, Oxalo
succinate, which remains enzyme-bound and undergoes
decarboxylation to α -ketoglutarate.
• The decarboxylation requires Mg++ or Mn++ ions.
• There are three isoenzymes of isocitrate dehydrogenase.
• One, which uses NAD+, is found only in mitochondria.
• The other two use NADP+ and are found in
mitochondria and the cytosol.

15. Respiratory chain-linked oxidation of isocitrate
proceeds almost completely through the NAD+-
dependent enzyme.

16. Step-4-Formation of Succinyl Co A
(Oxidative decarboxylation)
• The conversion of isocitrate into
αketoglutarate is followed by a second
oxidative decarboxylation reaction, the
formation of Succinyl CoA from
αketoglutarate

17. • α-Ketoglutarate undergoes oxidative decarboxylation
in a reaction catalyzed by a multi-enzyme complex
similar to that involved in the oxidative
decarboxylation of pyruvate.
• The α--ketoglutarate dehydrogenase complex requires
the same cofactors as the pyruvate dehydrogenase
complex—thiamine diphosphate, lipoate, NAD+,
FAD, and CoA— and results in the formation of
succinyl-CoA.

18. Step-5- Formation of Succinate
(Substrate level phosphorylation)
• Succinyl CoA is an energy-rich thioester
compound
• The cleavage of the thioester bond of succinyl
CoA is coupled to the phosphorylation of a
purine nucleoside diphosphate, usually GDP.
• This reaction is catalyzed by succinyl CoA
synthetase (succinate thiokinase).

19. This is the only example in the citric acid cycle of
substrate level phosphorylation.
Tissues in which gluconeogenesis occurs (the liver
and kidney) contain two isoenzymes of succinate
thiokinase, one specific for GDP

20. REACTIONS 6,7,8

21. Step-6- Formation of Fumarate
• The first dehydrogenation reaction, forming
fumarate, is catalyzed by succinate
dehydrogenase, which is bound to the inner
surface of the inner mitochondrial membrane.
E-FAD+Succinate E-FADH2+Fumerate
Step-7- Formation of Malate
• Fumarase (fumarate hydratase) catalyzes the
addition of water across the double bond of
fumarate, yielding malate.

22. Step-8- Regeneration of
oxaloacetate
• Malate is converted to oxaloacetate by malate
dehydrogenase, a reaction requiring NAD+.
• Although the equilibrium of this reaction
strongly favors malate, the net flux is to
oxaloacetate because of the continual removal of
oxaloacetate (to form citrate, as a substrate for
gluconeogenesis, or to undergo transamination to
aspartate) and also the continual reoxidation of
NADH.

23. Energy yield per Acetyl co A per
turn of 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 3, and 2 ATP of FADH2.
• Consequently, 11 high-transfer-potential phosphoryl groups
are generated when the electron-transport chain oxidizes 3
molecules of NADH and 1 molecule of FADH2,
• In addition, 1 ATP (or GTP) is formed by substrate-level
phosphorylation catalyzed by succinate thiokinase.

24. Energy yield per Acetyl co A per
turn of cycle
• 1 acetate unit generates approximately 12
molecules of ATP.
• In dramatic contrast, only 2 molecules of ATP are
generated per molecule of glucose (which generates
2 molecules of acetyl CoA) by anaerobic glycolysis.
• Molecular oxygen does not participate directly in
the citric acid cycle.
• However, the cycle operates only under aerobic
conditions because NAD+ and FAD can be
regenerated in the mitochondrion only by the
transfer of electrons to molecular oxygen.

25. SUMMARY OF TCA CYCLE

26. Significance of TCA cycle
• Complete oxidation of acetyl CoA.
• ATP generation.
• Final common oxidative pathway.
• Integration of major metabolic pathways.
• Fat is burned on the wick of carbohydrates.
• Excess carbohydrates are converted as neutral fat
• No net synthesis of carbohydrates from fat.
• Carbon skeleton of amino acids finally enter the
TCA cycle.

27. Energetics of TCA Cycle
• Oxidation of 3 NADH by ETC coupled with
oxidative phosphorylation results in the
synthesis of 9ATP.
• FADH2 leads to the formation of 2ATP.
• One substrate level phosphorylation.
• Thus, a total of 12 ATP are produced from one
acetyl CoA.

28. Regulation of TCA Cycle
• Three regulatory enzymes
1. Citrate synthase
2. Isocitrate dehydrogenase
3.α-ketoglutarate dehydrogenase

29. • Citrate synthase is inhibited by ATP, NADH, acyl
CoA& succinyl CoA.
• Isocitrate dehydrogenase is activated by ADP&
inhibited by ATP and NADH
• α-ketoglutarate dehydrogenase is inhibited by
succinyl CoA & NADH.
• Availability of ADP is very important for TCA cycle
to proceed.

30. Inhibitors of TCA Cycle
• Aconitase is inhibited by fluoro-acetate.
• This is a non-competitive inhibition.
• Alpha ketoglutarate is inhibited by Arsenite.
• This is also a non-competitive.
• Succinate dehydrogenase is inhibited by
malonate.
• This is competitive inhibition.

31. Amphibolic nature of the TCA
cycle
• TCA cycle is both catabolic & anabolic in
nature, called as amphibolic.
• Since various compounds enter into or leave
from TCA cycle, it is sometimes called as
metabolic traffic circle.

32. Important anabolic reactions of
TCA cycle
• Oxaloacetate is precursor for aspartate.
• α-ketoglutarate can be transaminated to
glutamate.
• Succinyl CoAis used for synthesis of heme.
• Mitochondrial citrate is transported to
cytoplasm & it is cleaved into acetyl CoA to
provide

33. Anaplerosis or anaplerotic
reactions
• The reactions concerned to replenish or to fill
up the intermediates of citric acid cycle are
called anaplerotic reactions or Anaplerosis.
• Pyruvate carboxylase catalyses conversion of
pyruvate to oxaloacetate.
• This is an ATP dependent carboxylation
reaction.

34. Example: pyruvate carboxylase, which uses a biotin
(vitamin B7) cofactor to carry CO2.
Transamination
Transamination is a process where an amino acid
transfers its amino group to a keto group and itself gets
converted to a keto acid.
The formation of Alpha ketoglutarate & oxaloacetate
occures by this mechanism.

35. THANK YOU