Prof. Dr.

MAHA SMAISM
Professor of clinical biochemistry
Department of Clinical Biochemistry
Faculty Of Medicine- University Of Babylon
 The Citric Acid Cycle
The citric acid cycle is a series of
reactions that connects the
intermediate acetyl CoA from the
catabolic pathways in stage 2 with
electron transport and the synthesis
of ATP in stage 3.
Learning Goal Describe the oxidation
of acetyl CoA in the citric acid cycle.
 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.
 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.
• PDH is subject to regulation by allosteric
mechanisms and covalent modification.
Allosteric inhibitors are the products acetyl PDH
+ATP
CoA and NADH.
• PDH is covalently modified by
phosphorylation by PDH kinase and
dephosphorylated by PDH phosphatase. The
dephosphorylated form of the enzyme is active.
Hence, activators of PDH kinase like ATP, Mg+2
AMP
NADH and acetyl CoA inhibit PDH reaction.
PDH phosphatase is activated by Ca++, Mg++
and AMP; this will increase the rate of PDH PDH-P

reaction whereas PDH kinase is inhibited by

Ca++. When there is adequate ATP and acetyl PDH kinase=PDKs
CoA, the enzyme is inhibited. PDH phosphatase=PDPs
 Citric Acid Cycle Overview
• In the citric acid cycle, eight reactions
oxidize acetyl CoA from pyruvate or fatty
acids, producing CO2 and the high-
energy compounds FADH2, NADH, and
GTP.
• Reactions involved in the citric acid cycle
include condensation, dehydration,
hydration, oxidation, reduction, and
hydrolysis.
 Reaction 1: Formation of Citrate
1. Condensation (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 allosterically activated by Ca2 + and ADP, and
inhibited by ATP, NADH, succinyl CoA, and fatty acyl CoA derivatives. However,
the primary mode of regulation is also determined by the availability of its
substrates,acetyl CoA and oxaloacetate. [Note: 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 (PFK), the rate-
limiting enzyme of glycolysis,and activates acetyl CoA carboxylase (the rate-limiting
enzyme of fatty acid synthesis)]
In the first reaction of the citric acid cycle,
• citrate synthase catalyzes the condensation of an acetyl
group (2C) from acetyl CoA with oxaloacetate (4C) to yield
citrate (6C) and coenzyme A.
• the energy to form citrate is provided by the hydrolysis of
the high-energy thioester bond in acetyl CoA.
 Reaction 2: Isomerization
Isomerization of citrate
citrate rearranges to isocitrate, a secondary alcohol.
• aconitase catalyzes the dehydration of citrate (tertiary alcohol) to
yield cis-aconitate, followed by a hydration that forms isocitrate
(secondary alcohol).
 Reaction 3: Oxidation, Decarboxylation
• 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 Ca++, and is
inhibited by ATP and NADH, whose levels are elevated when the cell
has abundant energy stores.
 Reaction 4: Decarboxylation, Oxidation
Oxidative decarboxylation of α-ketoglutarate
The conversion of α-ketoglutarate to succinyl CoA is catalyzed by the α-
ketoglutarate dehydrogenase complex. 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 coenzyme A. a-Ketoglutarate dehydrogenase
complex is inhibited by ATP, GTP, NADH, and succinyl CoA, and activated by
Ca++..
 Reaction 5: Hydrolysis
Cleavage of succinyl CoA
Succinate thiokinase (also called succinyl CoA synthetase)
cleaves the high-energy thioester bond of succinyl CoA(C-SCoA).
This reaction is coupled to phosphorylation of GDP to GTP. (GTP
and ATP are energetically interconvertible).The generation of
GTP by succinate thiokinase is another example of substrate-level
phosphorylation.
 Reaction 6: Hydrolysis
Succinate is oxidized to fumarate by succinate dehydrogenase,
producing the reduced coenzyme FADH .Succinate dehydrogenase
is inhibited by oxaloacetate.
 Reaction 7: Hydration

Hydration of fumarate
Fumarate is hydrated to malate in a freely reversible reaction
catalyzed by fumarase (also called fumarate hydratase).
 Reaction 8: Oxidation
In reaction 8, catalyzed by malate dehydrogenase,
• the hydroxyl group in malate is oxidized to a carbonyl group,
yielding oxaloacetate.
• oxidation provides hydrogen ions and electrons for the
reduction of NAD+ to NADH and H+.
 Summary, Citric Acid Cycle
In the citric acid cycle,
• an acetyl group bonds with oxaloacetate to form citrate.
• two decarboxylations remove two carbons as two CO2.
• four oxidations provide hydrogen for three NADH and one
FADH2.
• a direct phosphorylation forms GTP (ATP).
 Regulation of the Citric Acid Cycle

A. Regulation by activation and

inhibition of enzyme activities
TCA cycle is controlled by the
regulation of several enzyme
activities . The most important of
these regulated enzymes are
citrate synthase, isocitrate
dehydrogenase, and ketoglutarate
dehydrogenase complex.
B. Regulation by the availability of ADP
1. Effects of elevated ADP: Energy
consumption as a result of muscular
contraction, biosynthetic reactions, or other
results in the hydrolysis of ATP to processes
ADP and the resulting increase in the
concentration of ADP accelerates the rate of
reactions that use ADP to generate ATP.
Production of ATP increases until it matches
the rate of ATP consumption by
energy-requiring reactions which mean that
ADP activate TCA cycle
2. Effects of low ADP:
ADP (or Pi) is present in limiting concentration, the formation of ATP
by oxidative phosphorylation decreases as a result of the lack of
phosphate acceptor (ADP) or inorganic phosphate.
The rate of oxidative phosphorylation is proportional to
[ADP][Pi]/[ATP]; this is known as respiratory control of energy
production. The oxidation of NADH and FADH2 by the electron
transport chain also stops if ADP is limiting. This is because the
processes of oxidation and phosphorylation are tightly coupled and
occur simultaneously. So as NADH and FADH2 accumulate, their
oxidized forms become depleted, causing inhibition of TCA cycle.
AMPHIBOLIC PATHWAY
All pathways such as beta oxidation of fat or glycogen synthesis are
either catabolic or anabolic. But TCA cycle is truly amphibolic (both
catabolic and anabolic) in nature. Citric Acid Cycle components are
important biosynthetic intermediates in aerobic organisms. Besides
its role in the oxidative catabolism of carbohydrates, fatty acids, and
amino acids, the cycle provides precursors for many biosynthetic
pathways. Ketoglutarate and oxaloacetate can, for example, serve
as precursors of the amino acids aspartate and glutamate by simple
transamination which are then used to build other amino acids, as
well as purine and pyrimidine nucleotides, Oxaloacetate is converted
to glucose in gluconeogenesis, SuccinylCoA is a central intermediate
in the synthesis of the porphyrin ring of heme groups, which serve as
oxygen carriers (in hemoglobin and myoglobin) and electron carriers
(in cytochromes).
Influx of TCA cycle intermediates Efflux of TCA cycle intermediates
Anaplerotic Reactions
Anaplerotic Reactions replenish Citric Acid Cycle Intermediates as intermediates of
the citric acid cycle are removed to serve as biosynthetic precursors; they are
replenished by anaplerotic reactions. Under normal circumstances, the reactions by
which cycle intermediates are siphoned off into other pathways and those by
which they are replenished are in dynamic balance, so that the concentrations of
the citric acid cycle intermediates remain almost constant.
 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.
 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.
 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.