TCA CYCLE

1
 Introduction
• T h e citric acid cycle is the 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.
• I n eukaryotes, the reactions of the citric acid cycle take place inside
mitochondria, in contrast with those of glycolysis, which take place in
the cytosol.
 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 two molecules of
carbon dioxide and reduce the coenzymes that are re oxidized
through the electron transport chain, linked to the formation of
ATP.

3
 Overview of the Citric Acid
Cycle
•A four- carbon compound (oxaloacetate) condenses with a two-
carbon acetyl unit to yield a six-carbon tricarboxylic acid
(citrate).
•An isomer of citrate is then oxidatively decarboxylated.
•The resulting five-carbon compound (α-ketoglutarate) also is
oxidatively decarboxylated to yield a four carbon compound
(succinate).
Oxaloacetate is then regenerated from succinate.
•
Two carbon atoms enter the cycle as an acetyl unit and two carbon
•
atoms leave the cycle in the form of two molecules of carbon
dioxide.

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Overview of the Citric Acid
Cycle
o Three hydride ions (hence, six
electrons) are transferred to three
molecules of nicotinamide adenine
dinucleotide (NAD+), whereas one
pair of hydrogen atoms (hence,
two electrons) are transferred to
one molecule of flavin adenine
dinucleotide (FAD) .
o The function of the citric acid
cycle is the harvesting of high-
energy electrons from carbon
fuels.

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Citric acid cycle and requirement
of oxygen

Oxygen is required for the citric acid cycle indirectly in as

much as it is the electron acceptor at the end of the
electron- transport chain, necessary to regenerate NAD+
and FAD.
6
Citric acid cycle & requirement
of oxygen
o The citric acid cycle itself neither generates a large amount of ATP
nor includes oxygen as a reactant.
o Instead, the citric acid cycle removes electrons from acetyl
CoA and uses these electrons to form NADH and FADH2 .
o In oxidative phosphorylation, electrons released in the
reoxidation of NADH and FADH2 flow through a series of
membrane proteins (referred to as the electron-transport
chain) to generate a proton gradient across the membrane
o The citric acid cycle, in conjunction with oxidative
phosphorylation, provides the vast majority of energy used by
aerobic cells in human beings, greater than 95%.

7
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, one molecule of oxaloacetate is capable of participating
in the oxidation of many acetyl molecules

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 Reactions of the Citric Acid
Cycle
• Step-1 Formation of Citrate
• The citric acid cycle begins with the condensation of a four-
carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group
of acetyl CoA. Oxaloacetate 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.

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 Step-1-Formation of
Citrate

Oxaloacetate first condenses with acetyl CoA to form citryl

CoA, which is then hydrolyzed to citrate and CoA.

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 Step-2-Formation of
Isocitrate
• Citrate is isomerized into isocitrate to enable the six-carbon 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

11
.
Step-2-Formation of Isocitrate
(contd.)

Aconitase is an iron-sulfur protein, or nonheme iron protein.

It contains four iron atoms that are not incorporated as part of a
heme group.
12
 Step-2-Formation of Isocitrate
(contd.)
• The poison Fluoroacetate is toxic, because fluoroacetyl-CoA
condenses with oxaloacetate to form fluorocitrate, which inhibits
Aconitase, causing citrate to accumulate.
• The mode of inhibition is suicidal inhibition

13
 Step-3- Formation of α- Keto
Glutarate
• 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. 14
 Step-3- Formation of α- Keto
Glutarate (contd.)

Respiratory chain-linked oxidation of isocitrate

proceeds almost completely through the NAD+-
dependent enzyme.

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Step-4-Formation of Succinyl Co
A

The conversion of isocitrate into α- ketoglutarate is

followed by a second oxidative decarboxylation reaction,
the formation of Succinyl CoA from α- ketoglutarate.

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 Step-4-Formation of
Succinyl Co A
o α-Ketoglutarate undergoes oxidative decarboxylation in a reaction
catalyzed by a multi-enzyme complex similar to that involved in
the oxidative decarboxylation of pyruvate.
o 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.

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Step-4-Formation of Succinyl Co
A
• The equilibrium of this reaction is so much in favor of succinyl-
CoA formation that it must be considered to be physiologically
unidirectional.
• As in the case of pyruvate oxidation, arsenite inhibits the
reaction, causing the substrate, α-ketoglutarate, to accumulate.

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 Step-5- Formation of
Succinate
• 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).

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 Step-5- Formation of
Succinate

o This is the only example in the citric acid cycle of substrate

level phosphorylation.
o Tissues in which gluconeogenesis occurs (the liver and
kidney) contain two isoenzymes of succinate thiokinase, one
specific for GDP and the other for ADP

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 Step-5- Formation of
Succinate
• The GTP formed is used for the decarboxylation of oxaloacetate to
phosphoenolpyruvate in gluconeogenesis, and provides a regulatory
link between citric acid cycle activity and the withdrawal of
oxaloacetate for gluconeogenesis.
Non gluconeogenic tissues have only the isoenzyme that uses
ADP.

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 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.
• The enzyme contains FAD and iron-sulfur (Fe:S) protein,
and directly reduces ubiquinone in the electron
transport chain.

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Step-7- Formation of
Malate
Fumarase (fumarate
hydratase)
catalyzes the
addition of water
across the double
bond of fumarate,
yielding malate.

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 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 continuous reoxidation of
NADH

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Step-8- Regeneration of
oxaloacetate

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 TCA CYCLE

01/ 21/ 14 Biochemistry for medics

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 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.
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 Continue . . .
• 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. 28
 Regulation of the TCA
cycle
• Regulation of the TCA cycle like that of
glycolysis occurs at both the level of entry
of substrates into the cycle as well as at
the key reactions of the cycle.
• Fuel enters the TCA cycle primarily as
acetyl- CoA. The generation of acetyl-CoA
from carbohydrates is, therefore, a major
control point of the cycle.
• This is the reaction catalyzed by the
PDH complex. 29
 Regulation of the TCA cycle
1) Regulation of PDH Complex
a) Allosteric modification-PDH complex is inhibited by
acetyl- CoA and NADH and activated by non-acetylated
CoA (CoASH) and NAD+.
b) 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
.
 Regulation of the TCA cycle
(contd.)
2) 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 muscular
contraction and secretion, when there is
increased energy demand.
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 Regulation of TCA cycle
enzymes
a)Citrate synthase- There is allosteric inhibition
of citrate synthase by 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 inhibits iso-
citrate dehydrogenase by directly
displacing NAD+. ATP, too, is inhibitory.
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 Regulation of TCA cycle
enzymes
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.

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 TCA cycle and cellular ratio of
NAD+/NADH
• Since three reactions of the TCA cycle as well as PDH utilize
NAD+as co-factor, the cellular ratio of NAD+/NADH has a
major impact on the flux of carbon through the TCA cycle.
 • The activity of TCA cycle is immediately dependent on the

supply of NAD+, which in turn, because of the tight

coupling between oxidation and phosphorylation, is
dependent on the availability of ADP and hence, ultimately
on the rate of utilization of ATP in chemical and physical
• work.
 Thus, respiratory control via the respiratory chain and

oxidative phosphorylation primarily regulates citric acid

cycle activity.

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Regulation of TCA cycle-
Summary
Excess of ATP
depicts energy
rich state of the
cell, hence TCA
cycle is inhibited
while reverse
occurs when the
cell is in a low
energy state with
excess of ADP.
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 Significance of TCA
Cycle
• 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. 36
 A) 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 acetate unit generates approximately
12 molecules of ATP per turn of the
37
 B) Anabolic role of TCA
cycle
 As a major metabolic hub of the cell, the
citric acid cycle also provides intermediates
for biosynthesis of various compounds.
• i) Role in Gluconeogenesis- All the
intermediates of the cycle are potentially
glucogenic, since they can give rise to
oxaloacetate, and hence net production of
glucose (in the liver and kidney, the organs
that carry out gluconeogenesis.
38
 B) Anabolic role of TCA
cycle
Role in
gluconeogenesis

• The key enzyme that catalyzes net transfer out of

the cycle into gluconeogenesis is phospho-enol-
pyruvate carboxy kinase, which catalyzes the
decarboxylation of oxaloacetate to
phosphoenolpyruvate, with GTP acting as the
phosphate donor. 39
 ii) Role in synthesis of Non-
essential amino acids
Since the transamination reactions are reversible, the cycle
also serves as a source of carbon skeletons for the
synthesis of some amino acids like Alanine, aspartate,
Asparagine Glutamate , glutamine etc.

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Role of TCA cycle in the synthesis
of amino acids
Role of TCA cycle in the
synthesis of amino
acids

Aspartic acid is a precursor of Asparagine, Lysine, Methionine,

Threonine and Isoleucine. These amino acid except Asparagine are
essential amino acids, they are synthesized only in plants.
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 Role of TCA cycle in fatty acid
synthesis
 iii) Role in 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 is made available in the cytosol from citrate synthesized in the
mitochondrion, transported into the cytosol, and cleaved in a reaction
catalyzed by ATP-citrate lyase.

42
 Role of TCA cycle in fatty acid
synthesis
• Citrate is transported
out of the
mitochondrion when
Aconitase is
saturated with its
• substrate.
This ensures that
citrate is used for
fatty acid synthesis
only when there is
Acetyl co A can also be used for an adequate
the synthesis of cholesterol, amount to ensure
steroids etc. continued activity of
the cycle. 43
iv) Role in Heme
synthesis
• Succinyl co A
condenses with
amino acid
Glycine to form
Alpha amino
beta keto Adipic
acid, which is
the first step of
haem
biosynthesis. 44
v) Role in purine and pyrimidine
synthesis

Glutamate and Aspartate derived from TCA

cycle are utilized for the synthesis of
purines and pyrimidines.
45
 Role of vitamins
•
Riboflavin, in the form of flavin adenine dinucleotide (FAD),
a cofactor for succinate dehydrogenase
• Niacin, in the form of nicotinamide adenine
dinucleotide (NAD), the electron acceptor for
isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, and malate dehydrogenase;
• Thiamine(vitamin B1) , as thiamine pyro phosphate, the
coenzyme for decarboxylation in the α
-ketoglutarate dehydrogenase reaction;
Pantothenic acid, as part of coenzyme A such as acetyl-
• CoA and succinyl-CoA and
Biotin- in CO2 fixation reaction to compensate
oxaloacetate concentration
 Anaplerotic
•
reactions
Anaplerosis is the act of replenishing TCA cycle
intermediates that have been extracted for biosynthesis
(in what are called cataplerotic reactions).
• The TCA Cycle is a hub of metabolism, with central
importance in both energy production and biosynthesis.
Therefore, it is crucial for the cell to regulate
concentrations of TCA Cycle metabolites in the
• mitochondria.
Anaplerotic flux must balance cataplerotic flux in order to retain
homeostasis of cellular metabolism
•

47
 Anaplerotic
reactions
1) Formation of oxaloacetate from pyruvate
• In case oxaloacetate is converted into amino acids
for protein synthesis or used for gluconeogenesis
and, subsequently, the energy needs of the cell
• rise.
The citric acid cycle will operate to a reduced
extent unless new oxaloacetate is formed,
because acetyl CoA cannot enter the cycle unless
• it condenses with oxaloacetate.
Even though oxaloacetate is recycled, a minimal
level must be maintained to allow the cycle to
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Anaplerotic
reactions
Oxaloacetate is formed
by –
a)Carboxylation of
pyruvate, by pyruvate
carboxylase
b)Through formation of
malate from pyruvate by
Malic enzyme
c)From malate to
oxaloacetate by
malate
dehydrogenase

49
 Anaplerotic
reactions
2) Formation of oxaloacetate from Aspartate-
Oxaloacetate can also be formed from Aspartate
by transamination reaction.
3) Formation of Alpha keto glutarate- Alpha
ketoglutarate can be formed from Glutamate
dehydrogenase or from transamination
reactions.
4) Formation of Succinyl co A – Succinyl co A can
be produced from the oxidation of odd chain
fatty acid and from the metabolism of methionine
and isoleucine (through carboxylation of Propionyl 50
 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 co A represents fat component,
since the major source is fatty acid
oxidation.
• Acetyl co A is completely oxidized in the
TCA cycle in the presence of oxaloacetate.

51
 Fats burn in the flame of
carbohydrates?
• 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.
52