Krebs Cycle

KREBS CYCLE
Erdinç DEVRİM, MD
Professor of Medical Biochemistry
[email protected]
2 BIOMEDICAL IMPORTANCE
 The tricarboxylic acid cycle (the TCA cycle, also called the Krebs cycle or
the citric acid cycle) plays several roles in metabolism.
 The TCA 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.
 This oxidation provides energy for the production of the majority of ATP in
most animals, including humans.
 It also has a central role in gluconeogenesis, lipogenesis, and
interconversion of amino acids.
 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.
3 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.
 Two carbons entering the cycle as acetyl CoA are balanced by two CO2
exiting.
THE TCA CYCLE
From Essentials of Medical Biochemistry: With Clinical Cases, Second Edition.

Copyright © 2015 Elsevier Inc. All rights reserved.
5 Oxidative decarboxylation of pyruvate
 Pyruvate, the end product of aerobic glycolysis, must be

transported into the mitochondrion before it can enter the TCA
cycle.
 This is accomplished by a specific pyruvate transporter that helps
pyruvate cross the inner mitochondrial membrane.
 Once in the matrix, pyruvate is converted to acetyl CoA by the
pyruvate dehydrogenase complex, which is a multienzyme
complex.
 The pyruvate dehydrogenase complex is not part of the TCA cycle,
but is a major source of acetyl CoA which is substrate for the cycle.
6 Oxidative decarboxylation of pyruvate
 The pyruvate dehydrogenase complex (PDH complex) is a

multimolecular aggregate of three enzymes, pyruvate dehydrogenase
(PDH or E1, also called a decarboxylase), dihydrolipoyl transacetylase
(E2), and dihydrolipoyl dehydrogenase(E3).
 In addition to the enzymes participating in the conversion of pyruvate
to acetyl CoA, the complex also contains two tightly bound regulatory
enzymes, pyruvate dehydrogenase kinase and pyruvate
dehydrogenase phosphatase.
 The PDH complex contains five coenzymes that act as carriers or
oxidants for the intermediates of the reactions. E1 requires thiamine
pyrophosphate (TPP), E2 requires lipoic acid and CoA, and E3 requires
FAD and NAD+.
7 Regulation of the PDH complex
 Covalent modification by the two regulatory enzymes that

are part of the complex alternately activate and inactivate
E1 (PDH).
 The cyclic AMP-independent PDH kinase phosphorylates and,
thereby, inhibits E1, whereas PDH phosphatase
dephosphorylates and activates E1.
 Pyruvate is a potent inhibitor of PDH kinase.
 Although covalent regulation by the kinase and phosphatase
is main, the complex is also subject to product (NADH, acetyl
CoA) inhibition.
8 Synthesis of citrate from acetyl CoA
and oxaloacetate
 The condensation of acetyl CoA and oxaloacetate to form citrate (a
tricarboxylic acid) is catalyzed by citrate synthase.
 It is inhibited by its product, citrate.
 Substrate availability is another means of regulation for citrate synthase.
9 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.
10 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 ADP (a low-energy
signal) and Ca2+, and is inhibited by ATP and NADH.
11 Oxidative decarboxylation of
α-ketoglutarate
 The conversion of α-ketoglutarate to succinyl CoA is
catalyzed by the α-ketoglutarate dehydrogenase complex,
a multimolecular aggregate of three enzymes.
 The reaction releases the second CO2 and produces the
second NADH of the cycle.
 The coenzymes for the enzyme complex are thiamine
pyrophosphate, lipoic acid, FAD, NAD+, and CoA.
12 Cleavage of succinyl CoA
 Succinate thiokinase (also called succinyl CoA

synthetase) cleaves the high-energy thioester
bond of succinyl CoA.
 This reaction is coupled to phosphorylation of
guanosine diphosphate (GDP) to guanosine
triphosphate (GTP).
 The generation of GTP by succinate thiokinase is
another example of substrate-level
phosphorylation.
13 Oxidation of succinate
 Succinate is oxidized to fumarate by succinate

dehydrogenase, as FAD is reduced to FADH2.
 The reaction is inhibited by malonate.
 Succinate dehydrogenase is the only enzyme of the
TCA cycle that is embedded in the inner
mitochondrial membrane.
 It functions as Complex II of the electron transport
chain.
14 Hydration of fumarate
 Fumarate is hydrated to malate in a freely

reversible reaction catalyzed by fumarase
(also called fumarate hydratase).
 Fumarate is also produced by the urea cycle,
in purine synthesis, and during catabolism of
the amino acids, phenylalanine and tyrosine.
15 Oxidation of malate
 Malate is oxidized to oxaloacetate by malate dehydrogenase.

 This reaction produces the third and last 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
aspartic acid.
16 ENERGY PRODUCED BY THE TCA CYCLE
 Ten ATP are formed per turn of the citric acid 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 ∼2.5 ATP,
and of each FADH2 results in formation of ∼1.5 ATP.
 In addition, 1 ATP (or GTP) is formed by substrate-level
phosphorylation catalyzed by succinate thiokinase.
17 REGULATION OF THE TCA CYCLE
 The 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 surge in
ADP.
18 THE CITRIC ACID CYCLE PLAYS A
CRUCIAL ROLE IN METABOLISM
 The citric acid cycle is not only a pathway for oxidation of two
carbon units, but it is also a major pathway for
 interconversion of metabolites arising from transamination and
deamination of amino acids,
 providing the substrates for amino acid synthesis by transamination,
 providing the substrates for gluconeogenesis and fatty acid synthesis.
 Because it functions in both oxidative and synthetic processes, it is
amphibolic.
19 REFERENCES
 Lippincott’s Illustrated Reviews Biochemistry, 5th Edition. Harvey RA,

Ferrier DR. Lippincott Williams & Wilkins, 2011; Chapter 9.
 Harper’s Illustrated Biochemistry, 30th Edition. Rodwell VW, Bender
DA, Botham KM, Kennely PJ, Weil PA. Lange, 2015; Chapter 16&17.

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KREBS CYCLE

From Essentials of Medical Biochemistry: With Clinical Cases, Second Edition.

 Pyruvate, the end product of aerobic glycolysis, must be

 The pyruvate dehydrogenase complex (PDH complex) is a

 Covalent modification by the two regulatory enzymes that

 Citrate is isomerized to isocitrate by aconitase, an Fe-S protein.

 Succinate thiokinase (also called succinyl CoA

 Succinate is oxidized to fumarate by succinate

 Fumarate is hydrated to malate in a freely

 Malate is oxidized to oxaloacetate by malate dehydrogenase.

 The TCA cycle is controlled by the regulation of several enzyme

 Lippincott’s Illustrated Reviews Biochemistry, 5th Edition. Harvey RA,

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