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Tricarboxylic Acid Cycle

Dr Tolunimi Adedeji (MD)

I. Overview

The tricarboxylic acid cycle or TCA cycle, also known as the Krebs
cycle or citric acid cycle, is an important component in metabolism
It is the converging point where oxidative metabolism of
carbohydrates, amino acids, and fatty acids occurs
These compounds are converted to CO2 and H2O
This oxidation facilitates the production of ATP, thus, an energy source
in most animals
TCA cycle occurs exclusively in the mitochondria and is in close
proximity to reactions of electron transport
The cycle is aerobic in nature (O2 required as final electron acceptor)
TCA cycle also involved in the formation of glucose from some amino
acids and in formation of building blocks for synthesis of some amino
acids and heme
Intermediates of the cycle can also be produced via catabolism of
some amino acids

II. Reactions of the TCA cycle

Oxaloacetate first undergoes


condensation upon reaction with
an acetyl group of acetyl CoA
Oxaloacetate is produced once
more upon completion of the
cycle

II. Reactions of the TCA cycle


A.

Oxidative decarboxylation
of pyruvate

Pyruvate (product of
aerobic glycolysis) is taken
to the mitochondrion prior
to decarboxylation by a
pyruvate transporter
Pyruvate then converted to
acetyl CoA by pyruvate
dehydrogenase complex
(multienzyme complex)
see figure

II. Reactions of the TCA cycle

The pyruvate dehydrogenase complex consists of three enzymes:


1.
2.
3.

pyruvate dehydrogenase (E1)


dihydrolipoyl transacetylase (E2)
dihydrolipoyl dehydrogenase (E3)

The complex also contains five coenzymes that serve as carriers


or oxidants for the intermediates of acetyl CoA (see figure on
next slide); coenzymes are:
1.
2.
3.
4.
5.

thiamine pyrophosphate
lipoic acid
coenzyme A
FAD
NAD+

II. Reactions of the TCA cycle


Mechanism of action of the pyruvate dehydrogenase complex

TPP = thiamine pyrophosphate; L = lipoic acid

II. Reactions of the TCA cycle


B.

Synthesis of citrate from acetyl


CoA and oxaloacetate

This aldol condensation reaction


requires the enzyme citrate
synthase (see figure)
The enzyme is allosterically
activated by Ca2+ and ADP, and
inhibited by ATP, NADH, succinyl
CoA, and fatty acyl CoA
derivatives
Citrate inhibits
phosphofructokinase, the ratedetermining enzyme of glycolysis
and activates acetyl CoA
carboxylase (rate-limiting
enzyme of fatty acid synthesis)

II. Reactions of the TCA cycle


C.

Isomerization of citrate

The enzyme aconitase facilitates the isomerization of


citrate to isocitrate (see previous figure)
The enzyme is inhibited by fluoroacetate a component
of rat poison
Fluoroacetate is converted to fluoroacetyl CoA, which
reacts with oxaloacetate to produce fluorocitrate; citrate
levels then rise

II. Reactions of the TCA cycle


D.

Oxidation and decarboxylation of isocitrate

Oxidative decarboxylation of isocitrate is carried out in


the presence of isocitrate dehydrogenase to give ketoglutarate, as well as one of the three NADH
molecules produced by the cycle, and a CO 2 molecule
(see figure)
This reaction is one of the rate-determining steps of the
cycle
Enzyme is allosterically activated by ADP and Ca2+, and
is inhibited by ATP and NADH

II. Reactions of the TCA cycle


E.

Oxidative decarboxylation of ketoglutarate


This reaction is achieved in the
presence of -ketoglutarate
dehydrogenase complex to produce
succinyl CoA see figure
This reaction releases the second CO2
and NADH molecules of the cycle
Coenzymes required are thiamine
pyrophosphate, lipoic acid, FAD,
NAD+, and coenzyme A
The enzyme is inhibited by ATP, GTP,
NADH, and succinyl CoA and activated
by Ca2+

II. Reactions of the TCA cycle


F.

Cleavage of succinyl CoA

The enzyme succinate thiokinase is responsible for the


cleavage of succinyl CoA to give succinate and CoA, see
previous figure
This reaction is coupled to phosphorylation of GDP to GTP

G.

Oxidation of succinate

Succinate is oxidized to fumarate by succinate


dehydrogenase to give the reduced coenzyme FADH 2
from FAD (see previous figure)
The enzyme is inhibited by oxaloacetate

II. Reactions of the TCA cycle


H.

Hydration of fumarate

Fumarate is hydrated to malate


by fumarase (see previous figure)
Fumarate is also produced by the
urea cycle, in purine synthesis,
and during catabolism of amino
acids (phenylalanine and tyrosine)

I.

Oxidation of malate

Malate is oxidized to
oxaloacetate in the presence of
malate dehydrogenase (see
figure)
Reaction affords the third and
final NADH of the cycle

III. Energy produced by the TCA


cycle

Total yield of ATP from the oxidation of one acetyl CoA


shown in figure 9.8 below
Three pairs of electrons transferred reducing NAD + to
NADH and one pair reducing FAD to FADH 2

IV. Regulation of the TCA cycle

regulation can occur by activation


and inhibition of enzyme activities
(see figure)
This is different to glycolysis, since
phosphofructokinase primarily
regulates glycolysis
The regulated enzymes include
citrate synthase, isocitrate
dehydrogenase, and ketoglutarate dehydrogenase
complex
Regulation may also arise by the
availability of ADP elevated ADP
activates TCA cycle; low levels of
ADP lowers activity of TCA cycle

Remember that 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 acetylCoA.
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.

By way of review, the PDH complex is inhibited by acetylCoA and NADH and activated by non-acetylated CoA
(CoASH) and NAD+.
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).
The phosphorylation of PDH inhibits its activity and,
therefore, leads to decreased oxidation of pyruvate. 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 Mg 2+ and
Ca2+.

Since three reactions of the TCA cycle as well as PDH


utilize NAD+ as co-factor it is not difficult to understand
why the cellular ratio of NAD+/NADH has a major impact
on the flux of carbon through the TCA cycle.
Substrate availability can also regulate TCA flux.
This occurs at the citrate synthase reaction as a result of
reduced availability of oxaloacetate.
Product inhibition also controls the TCA flux, e.g. citrate
inhibits citrate synthase, -KGDH is inhibited by NADH
and succinyl-CoA.
The key enzymes of the TCA cycle are also regulated
allosterically by Ca2+, ATP and ADP.

Key concept map for tricarboxylic


acid cycle

Any Questions?