TRICARBOXYLIC ACID CYCLE

By Kinyi H.

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 OVERVIEW
• It is the final pathway where the oxidative metabolism
of carbohydrates, amino acids, and fatty acids
converge, their carbon skeletons being converted to
CO2.
• This oxidation provides energy for the production of
the majority of ATP in most animals, including humans.
• The cycle occurs totally in the mitochondria and is,
therefore, in close proximity to the reactions of
electron transport , which oxidize the reduced
coenzymes produced by the cycle.
• The TCA cycle is an aerobic pathway, because O2 is
required as the final electron acceptor.

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 OVERVIEW
• Reactions such as the catabolism of some amino acids
generate intermediates of the cycle and are called
anaplerotic reactions.
• The citric acid cycle also participates in a number of
important synthetic reactions. For example, the cycle
functions in the formation of glucose from the carbon
skeletons of some amino acids, and it provides building
blocks for the synthesis of some amino acids and
heme.
• Therefore, this cycle should not be viewed as a closed
circle, but instead as a traffic circle with compounds
entering and leaving as required. (amphibolic)

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 Location of TCA
• Operates in the mitochondria of eukaryotes
and in the cytosol of prokaryotes.
• Succinate dehydrogenase is the only
membrane bound enzyme in the cycle.
• It is embedded in the inner mitochondrial
membrane in eukaryotes.

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 Reactions of the TCA Cycle
• In the TCA cycle, oxaloacetate is first
condensed with an acetyl group from acetyl
coenzyme A (CoA), and then is regenerated as
the cycle is completed.
• Thus, the entry of one acetyl CoA into one
round of the TCA cycle does not lead to the
net production or consumption of
intermediates.

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Pyruvic Acid to Acetyl CoA

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 Synthesis of citrate from acetyl CoA
and oxaloacetate
• The condensation of acetyl CoA and oxaloacetate to form
citrate is catalyzed by citrate synthase .
• Citrate synthase is inhibited by its product, citrate, and by
NADH and succinyl CoA.
• Substrate availability is a key means of regulation for citrate
synthase.
• Citrate, in addition to being an intermediate in the TCA
cycle, provides a source of acetyl CoA for the cytosolic
synthesis of fatty acids.
• Citrate also inhibits phosphofructokinase, the rate-setting
enzyme of glycolysis, and activates acetyl CoA carboxylase
(the rate-limiting enzyme of fatty acid synthesis)

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 Isomerization of citrate
• Citrate is isomerized to isocitrate by aconitase,
an Fe-S protein.
• Aconitase is inhibited by fluoroacetate, a
compound that is used as a rat poison.
• Fluoroacetate is converted to fluoroacetyl
CoA, which condenses with oxaloacetate to
form fluorocitrate—a potent inhibitor of
aconitase—resulting in citrate accumulation.

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 Oxidation and decarboxylation of
isocitrate
• Isocitrate dehydrogenase catalyzes the irreversible
oxidative decarboxylation of isocitrate, yielding the first
of three NADH molecules produced by the cycle, and
the first release of CO2.
• This is one of the rate-limiting steps of the TCA cycle.
The enzyme is allosterically activated by adenosine
diphosphate (ADP, a low-energy signal) and Ca2+, and is
inhibited by adenosine triphosphate (ATP) and NADH,
whose levels are elevated when the cell has abundant
energy stores.

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 Oxidative decarboxylation of α-
ketoglutarate
• The conversion of α-ketoglutarate to succinyl CoA is
catalyzed by the α-ketoglutarate dehydrogenase
complex, which consists of three enzymatic activities .
• The mechanism of this oxidative decarboxylation is
very similar to that used for the conversion of pyruvate
to acetyl CoA.
• The reaction releases the second CO2 and produces the
second NADH of the cycle.
• The coenzymes required are thiamine pyrophosphate,
lipoic acid, FAD, NAD+, and CoA. Each functions as part
of the catalytic mechanism in a way analogous to that
described for pyruvate dehydrogenase complex.

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 Oxidative decarboxylation of α-
ketoglutarate
• The equilibrium of the reaction is far in the
direction of succinyl CoA—a high-energy
thioester similar to acetyl CoA.
• α-Ketoglutarate dehydrogenase complex is
inhibited by ATP, GTP, NADH, and succinyl CoA,
and activated by Ca++.
• α-Ketoglutarate is also produced by the
oxidative deamination or transamination of
the amino acid, glutamate.

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 Cleavage of succinyl CoA
• Succinate thiokinase (also called succinyl CoA synthetase—
named for the reverse reaction) cleaves the high-energy
thioester bond of succinyl CoA.
• This reaction is coupled to phosphorylation of guanosine
diphosphate (GDP) to guanosine triphosphate (GTP).
• GTP and ATP are energetically interconvertible by the
nucleoside diphosphate kinase reaction.
• The generation of GTP by succinate thiokinase is another
example of substrate-level phosphorylation.
• Succinyl CoA is also produced from propionyl CoA derived
from the metabolism of fatty acids with an odd number of
carbon atoms, and from metabolism of several amino acids.
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 Oxidation of succinate
• Succinate is oxidized to fumarate by succinate
dehydrogenase, producing the reduced
coenzyme FADH2 .
• Succinate dehydrogenase is the only enzyme
of the TCA cycle that is embedded in the inner
mitochondrial membrane. As such, it
functions as Complex II of the electron
transport chain.

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 Hydration of fumarate
• Fumarate is hydrated to malate in a
freely reversible reaction catalyzed by
fumarase (fumarate hydratase).
• Fumarate is also produced by the urea
cycle, in purine synthesis, and during
catabolism of the amino acids,
phenylalanine and tyrosine .

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 Oxidation of malate
• Malate is oxidized to oxaloacetate by malate
dehydrogenase. This reaction produces the
third and final NADH of the cycle.
• The ΔG0 of the reaction is positive, but the
reaction is driven in the direction of
oxaloacetate by the highly exergonic citrate
synthase reaction.
• Oxaloacetate is also produced by the
transamination of the amino acid, aspartic
acid .

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 Energy Produced by the TCA Cycle
• Two carbon atoms enter the cycle as acetyl CoA and
leave as CO2.
• The cycle does not involve net consumption or
production of oxaloacetate or of any other
intermediate.
• Four pairs of electrons are transferred during one turn
of the cycle: three pairs of electrons reducing three
NAD+ to NADH and one pair reducing FAD to FADH2.
• Oxidation of one NADH by the electron transport chain
leads to formation of 2.5 ATP, whereas oxidation of
FADH2 yields 1.5 ATP.

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 Regulation of the TCA Cycle
• TCA cycle is controlled by the regulation of several
enzyme activities.
• The most important of these regulated enzymes are
those that catalyze reactions with highly negative ΔG0:
citrate synthase, isocitrate dehydrogenase, and α-
ketoglutarate dehydrogenase complex.
• Reducing equivalents needed for oxidative
phosphorylation are generated by the pyruvate
dehydrogenase complex and the TCA cycle, and both
processes are upregulated in response to a rise in ADP.

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ATP

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 Reactions that deplete TCA
• The intermediates of the TCA cycle serve as
precursors for a variety of different pathways present
in different cell types . This is particularly important
in the central metabolic role of the liver.
• After a high carbohydrate meal, citrate efflux and
cleavage to acetyl CoA provides acetyl units for
cytosolic fatty acid synthesis.
• During fasting, gluconeogenic precursors are
converted to malate, which leaves the mitochondria
for cytosolic gluconeogenesis.

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 Reactions that deplete TCA
• The liver also uses TCA cycle intermediates to
synthesize carbon skeletons of amino acids.
Succinyl CoA may be removed from the TCA cycle
to form heme in cells of the liver and bone
marrow.
• In the brain,α -ketoglutarate is converted to
glutamate and then to γ-aminobutyric acid
(GABA), a neurotransmitter.
• In skeletal muscle,α-ketoglutarate is converted to
glutamine, which is transported through the
blood to other tissues.

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 Anaplerotic Reactions
• As intermediates of the citric acid cycle are
removed to serve as biosynthetic precursors,
they are replenished by anaplerotic reactions.
• They include:
Amino acid Metabolism
Metabolism of odd chain fatty acids
Pyruvate carboxylase reaction

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Odd Chain Fatty Acids

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 PYRUVATE CARBOXYLASE
ENZYME
• It catalyzes the addition of CO2 to pyruvate to
form oxaloacetate . Contains biotin, which
forms a covalent intermediate with CO2 in a
reaction requiring ATP and Mg2.
• Pyruvate carboxylase is found in many tissues,
such as liver, brain, adipocytes, and
fibroblasts, where its function is anaplerotic.
• Pyruvate carboxylase is activated by acetyl
CoA and inhibited by high concentrations of
many acyl CoA derivatives.

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 AMINO ACID DEGRADATION
• Transaminases form α-ketoglutarate and
oxaloacetate, TCA intermediates.
• Glutamate dehydrogenase also produces α-
ketoglutarate
• Succinyl CoA is formed from isoleucine, valine,
methionine and threonine.

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• Arsenic poisoning is caused by the presence of a large
number of different arsenious compounds that are
effective metabolic inhibitors.
• Acute accidental or intentional arsenic poisoning
requires high doses and involves arsenate (AsO42) and
arsenite (AsO2).
• Arsenite, which is 10 times more toxic than arsenate,
binds to neighboring sulfhydryl groups, such as those in
dihydrolipoate and in nearby cysteine pairs (vicinal)
found in α- keto acid dehydrogenase complexes and in
succinic dehydrogenase.
• Arsenate weakly inhibits enzymatic reactions involving
phosphate, including the enzyme glyceraldehyde 3-P
dehydrogenase in glycolysis.

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 Revision Question
A male infant is born without complications and appears
normal until 1year of age, when he has trouble standing. By
age 2yrs, he exhibits severe ataxia and psychomotor
retardation. He cannot stand or walk and he speaks very
little. Urinalysis reveals high levels of alanine. Blood tests
revealed elevated levels of pyruvate and lactate.
Therapeutic doses of thiamine are administered and within
6 months the boy can walk and speak.
• What is the diagnosis?
• Why did therapeutic doses of thiamine correct this
problem?
• What is the long term prognosis?

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