Last Update: 3 November 2017 Part I

TCA CYCLE M - 94

Aerobic metabolism of carbohydrate is carried out in two phases. Pyruvate, generated from
glycolytic pathway is oxidatively decarboxylated to acetyl CoA. This activated acetyl unit is then
completely oxidized to CO2 by TCA cycl or citric acid cycle or Kreb cycle. The citric acid cycle is also the
final common pathway for the oxidation of amino acid, fatty acid etc.
This cycle also provides intermediates for bio-synthetic path way.
Citric acid cycle was postulated by Hans. Krebs in 1937. In
eukaryotes the reaction of the TCA cycle occurs inside the
mitochondria.
Two carbon atoms enter the cycle as an acetyl unit and two
carbon atoms leave the cycle in the form of two molecule of CO2.
three hydride ions (six electron) are transferred to three NAD+ and
one pair of hydrogen atoms is transferred to FAD molecule. These
carriers produce ATP when they are oxidized by oxygen in electron
transport chain. The over view of the cycle is like that
Fig An overview of the citric acid cycle.
FORMATION OF ACETYL-COA FROM PYRUVATE
The oxidative decarboxylation of pyruvate to form acetyl CoA occurs in the mitochondrial matrix. It
is the link between gylcolysis and TCA cycle. This irreversible reaction is catalyzed by a multienzyme
system, Called pyruvate
dehydrogenase complex.

Fig.- The overall reaction catalyzed

by the pyruvate dehydrogenase
complex. The five coenzymes
involved in this reaction, and the
three enzymes that make up the
enzyme complex, are discussed in
the text.
In this reaction three different enzymes E1, E2 and E3 as well as 5 diff. Co-enzymes TPP, FAD,
(Flavindinucleotide ) NAD, CoA and lipoate are involved. TPP, FAD and lipoate served as catalytic co-
factors and CoA and NAD+ are the stoichiometric cofactors.
Enzyme Co-enzyme Molecular weight Number of subunits Reaction catalysed
of subunit. per complex
Pyruvate TPP 96,000 24 Oxidative decarboxylation
dehydrogenase (E1) of pyruvate
Dihydrolipoyl lipoate, CoA 65,000-70,000 24 Transfer of the acetyl
transacetylase (E2) group to CoA
Dihydrolipoyl FAD, NAD 56,000 12 Regeneration of oxidized
dehydrogenase (E3) form of lipoamide

There are four step in conversation of pyruvate into acetyl CoA.

First pyruvate is decarboxylated after it combines with TPP. This reaction is catalyzed by E1. TPP is
first converted into a carbanion, then carbon group of pyruvate adds with TPP and ultimately forms
hydroxymethyl TPP. 2nd TPP is oxidized to form an acetyl group and concomitantly transferred to
lipoamide. Here the disulfide group of lipoamide is converted into sulfhahydril form. After reaction yield

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product is acetyl-lipoamide. In 3rd step the acetyl group from acetyl-lipoamide is transferred to CoA to form
acetyl CoA. This reaction is catalyzed by E2.
In last stage, the oxidized form of lipoamide is regenerated by E3. Here two electrons are transferred
to FAD and then to NAD+.

REACTION OF TCA CYCLE

In this reaction sequence 8
successive reactions are required for the
total cycle.
1) FORMATION OF CITRATE
Citrate synthase condenses
acetyl CoA with oxaloacetate to form
citrate. Citrate synthase is a
homodimeric enzyme. In this reaction
the methyl carbon of acetyl group is
joined to the ketonyl carbon of
oxaloacetate. Thus an enzyme citryl
CoA is formed as intermediate. The thioester bond is hydrolyzed to release citrate and coenzyme-A.
Citrate synthase is a dimer of identical 49 kd subunit and has large and small domain in each subunit.
Between each large and small domain active site present. Oxaloacetate first binds and induces a major
structural rearrangement leading to the creation of a binding site for acetyl CoA. After binding of
oxaloacetate the small domain rotated 19
relative to large domain. The large domain
also moves 15A by rotation of helix.

2) FORMATION OF ISOCITRATE
The enzyme aconitase catalyzes the reversible transformation of citrate to isocitrate.
At first dehydrates citrate to cis-aconitate by removing of H and OH groups from Methylene carbon
and ketonyl carbon of oxaloacetate. Cis aconitate does not dissociated from active site of enzyme. Thus
aconitase reversibly promotes the addition of H and OH group in a reverse order to those C. This enzyme is
a homodimeric protein contains iron-sulphur (Fe4S4) clascher at centre. This Fe4S4 acts as a substrate
binding site and participates in addition and removal of H2O.

3) OXIDATION OF ISOCITRATE TO -KETOGLUTARATE

Isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate to form ketoglutarate.
At first, isocitrate oxidizes to oxalosuccinate using NAD+ as co-factor, NAD+ accepts hidride ion from
isocitrate. Oxalosuccinate changes into 5-carbon -ketoglutarate by removing a CO2.

4) OXIDATION OF -KETOGLUTARATE TO SUCCINYL CoA

The next step is another oxidative decarboxylation in which -keto glutarate is converted to succinyl
CoA by the action of enzyme -ketoglutarate dehydrogenase complex. Here NAD+ is served as electron
acceptor. After decarboxylation 5-carbon-keto acid convert into 4-carbon- succinyl CoA, releasing of a
CO2. The energy of oxidation -ketoglutarate is conserved in the formation of the thioester bond of succinyl
CoA.
-ketoglutarate dehydragenase complex also has 3 enzyme E1, E2 and E3, analogous to pyruvate
dehydrogenase complex. E1 is -ketoglutarate dehydrogenase is same as E1 of pyruvate dehydrogenase
complex. E2 of -ketoglutarate dehydrogenase is not identical but similar with E2 components of pyruvate

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deaydrogenase complex. E3 -ketoglutarate dehydrogenase complex and E3 of pyruvate dehydrogenase
complex are identical. The co-factors of the enzymes are similar in both cases.

5) CONVERSATION OF SUCCINYL CoA TO SUCCINATE

Succinyl CoA synthetase / succinate thiokinase catalyzes the cleavage of the high energy thioester
bond of succinyl CoA to produce succinate and CoA. This reaction has negative free energy. This energy is
used to drive the synthesis of a phosphor-anhydride bond in GTP.
Succinyl CoA synthetase is a heterodimeric enzyme. During reaction the enzyme molecules become
phosphorylated at a histidine residue in its active site. This phosphate group which has a high group transfer
potential, is transferred to GDP to form GTP.

Figure : Intermediates in the

succinyl-CoA synthetase reaction.
In step (1) a phosphate group
replaces CoA in the enzyme-bound
succinyl-CoA, forming a high-
energy acyl phosphate. In step (2)
the succinyl phosphate donates its
phosphate group to a His residue on
the enzyme, forming the high
energy phosphohistidyl enzyme. In
step (3) the phosphate group is
transferred from the His residue to
the terminal phosphate of GDP,
forming GTP.

6) OXIDATION OF SUCCINATE TO FUMARATE

Succinate is oxidized to fumarates by flavoprotein succinate dehydrogenase. Succinate
dehydrogenase is a heterodimeric protein along with 3 iron-sulphur 12Fe-2S, 3Fe-2S, 4Fe-4S cluster and
one cytochrome b560. Succinate dehydrogenase transfers H+ and electron from succinate to FAD. Therefore
succinate oxidizes to fumarate & FAD reduces to FADH2 reoxydized by transferring electron in electron
transport chain.

7) HYDRATION OF FUMARATE TO PRODUCE MALATE

The enzyme fumarase also a homodimeric protein reversibly hydrates fumarate to malate. This
enzyme is highly specific for trans isomer fumarate, and not its cis-isomer malate.

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8) OXIDATION OF MALATE TO OXALOACETATE
In the last reaction of TCA cycle, NAD linked malate dehydrogenase catalyzes the oxidation of
malate to oxaloacetate. This enzyme also homodimeric sulfhydril is component. In cell oxaloacetate
continually removed by the citrate synthase reaction. Thus the concentration of oxaloacetate is extremely
low cell which pulls the malate dehydrogenase reaction towards oxaloacetate formation.

ENERGETICS
The aerobic metabolism of pyruvate molecule gives 15 high energy phosphate bonds. 3 from
oxidative decarboxylation of pyruvate and 12 from TCA cycle steps. 3 NADH are produced from the cycle.
NADH transfer their electron in complex-I in the E.T.C. with the help of NADH Q- reductase to
ubiquinone of co-enzyme-Q. By this way 3 ATP are produced per NADH molecule. On the other hand FAD
transfer their electro directly in co-enzyme Q or ubiquinol through the complex II by the help of succinate
Q- reductase. Two high energy phosphate-band is generated from each FAD molecule. One GTP is
produced during the cyclic reaction without help of E.T.C. This is known as substrate level phosphorylation.
This total ATP production from aerobic metabolism of carbohydrates is given below

Relation Enzyme Mode of High-energy bonds

phosphorylation formed
Per C3 Per C6
Pyruvate acetyl CoA Pyruvate Oxidative 3 6
dehydrogenase
Isocitrate -ketoglutarate Isocitrate Oxidative 3 6
dehydrogenase
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-ketoglutarate succinyl. CoA -ketoglutarate Oxidative 3 6
dehydrogenase
Succinyl (CoA) Succinate Succinate thiokinase Substrate level 1 2
Succinate Fumarate Succinate Oxidative 2 4
dehydrogenase
Malate oxaloacetate Malate Oxidative 3 6
dehydrogenase
From Glycolysis 8
Total 38

REGULATION
Pyruvate dehydrogenase system is the key
regulatory multienzyme for regulation of TCA cycle.
Reversible covalent modification through
phosphorylation and dephosphorylation inactivate or
activate the enzyme respectively. The specific serine
residue of E1 is phosphorylated with help of pyruvate
dehydrogenase kinase which activate the enzyme.
Pyruvate dehydrogenase phosphatase
dephosphorylates the enzyme and converts into its
active form.
A rise in ATP/ADP ratio, NADH/NAD+ ratio, acetyl CoA concentration and cyclic AMP
concentration activates the PDH kinase which allosterically inactivates the PDH. During -oxydation supply
of acetyl CoA and ATP are sufficient. Therefore by inhibiting PHD through allosteric modification prevents
the break down of carbohydrate products.

Activator
In citric acid cycle -ketoglutarate dehydragenase, citrate synthase and isocitrate dehydrogenase are
the main rate limiting enzymes.
-keto glutarate dehydragenase is also regulated by allosterically. The regulation process is similar
as pyruvate dehydragenase system. Ca2+, ADP, AMP, also enhance the activity of -keto glutarate
dehydrogenase. But the other system like ADP/ATP ratio, NADH/NAD+ succinyl CoA ratio control the
activity of -keto glutarate dehydragenase complex as similar as pyruvate dehydrogenase complex system.

Citrate synthase is activated by acetyl CoA and oxaloacetate. Citrate competitively inhibits the citrate
synthase. Succinyl CoA also inhibits the citrate synthase. The regulation process of citrate synthase is like
that :-
Citrate synthase is a dimer and has large and small domain in each subunit. Between each large and
small domain active site is present. Oxaloacetate first binds and induces a major structural rearrangement
leading to creation of a binding site for acetyl-CoA. After binding of oxaloacetate, the small domain rotated
19 relative to large domain. The large domain also moves 15A by rotation of helix. Therefore
oxaloacetate helps the binding of acetyl group and their cooperative function controls its activity. The
activity of the citrate synthase is inhibited by ATP, NADH, acetyl-CoA etc.
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 Another regulatory enzyme is isocitrate dehydrogenase. It is activated by high concentration of Ca2+
and its substrate isocitrate, but inhibited by ATP and NADH ADP also activates the enzyme allosterically.
ADP decreases the Km value and increases the affinity of its substrate isocitrate.

Thus when ATP level decreases and ADP level increases the rate of TCA cycle rises by activation of
rate limiting enzyme.

ANABOLIC ROLE OF TCA CYCLE

The intermediates of the TCA cycle enter into the different anabolic reaction for synthesis of
porphyrins, fatty acid, gluconeogenesis, amino genesis etc.
a) Porphyrin Synthesis : Succinyl-CoA is used for synthesizing porphyrins.
b) Fatty acid synthesis : In hepatocytes, adipocytes and lactating mammocytes of humans and non
ruminant animals, citrate is transported from mitochondria to cytosol for fatty acid synthesis.
c) Cholesterol synthesis : In hepatocytes and other steroidogenic cells, citrate is also used for cholesterol
synthesis.
d) Gluconeogenesis : Malate and oxaloacetate are changed to glucose by gluconeogenesis.
e) Amino acid synthesis : -Keto glutarate, oxaloacetate may be transaminated by specific transaminase
into glutamate and aspartate.

Figure : Intermediates of
the citric acid cycle are
drawn off as precursors in
many biosynthetic
pathways, yielding the
products in the shaded
areas. Shown in red are
four anaplerotic reactions
that replenish depleted
intermediates of the citric
acid cycle.

Anaplerosis :
TCA cycle intermediates are continually removed by anabolic role of TCA cycle. Anaplerosis is the
generation of TCA cycle intermediates to keep the cycle operative. The following anaplerotic reactions
regenerates the intermediates.
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 a) HCO3-(CO2)+ATP + Pyruvate oxaloacetate pyruvate carboxylase + ADP + Pi
b) Glutamate+NADP++H2O ketogutarate + NH+4 + NADPH + H+ glutanale dehydrogenase
c) Glutamate + Pyruvate ketoglutarate + alamine. Transaminase

Figure : The structure of coenzyme A. A hydroxyl group of pantothenic acid is joined to a modified ADP moiety by a phosphate
ester bond, and its carboxyl group is attached to -mercaptoethylamine in amide linkage. The hydroxyl group at the 3 position of
the ADP moiety has a phosphate group not present in ADP itself. The-SH group of the mercaptoethylamine moiety forms a
thioester with acetate in acetyle-CoA (lower left). Coenzyme A is abbreviated as CoA, and acetyl coenzyme A as acetyl-CoA; the
reactive SH group is generally shown only in chemical structures.

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