Download THE CITRIC ACID CYCLE

The final common pathway for the oxidation of
fuel molecules., namely amino acids, fatty acids,
and carbohydrates
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In eukaryotes,
 Citric acid cycle inside
mitochondria, while
 Glycolysis in cytosol.
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Overview of the Citric Acid Cycle
 It is the gateway to the aerobic metabolism of any
molecule that can be transformed into an acetyl group
or dicarboxylic acid.
 The cycle is an important source of precursors:
 For the storage forms of fuels.
 For the building blocks of many other molecules such
as amino acids, nucleotide bases, and cholesterol.
 The citric acid cycle includes a series of redox reactions
that result in the oxidation of an acetyl group to two
molecules of CO2.
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The citric acid cycle is highly efficient:
 From a limited number of molecules a large amounts
of NADH and FADH2 are generated (account for >
95% of energy)
 An acetyl group (two-carbon units) is oxidized to:
1.
2.
3.
Two molecules of CO2
One molecule of GTP
High-energy electrons in
the form of NADH and
FADH2.
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Cellular Respiration
The citric acid cycle constitutes the first stage in cellular respiration,
the removal of high-energy electrons from carbon fuels.
These electrons reduce O2 to generate a
proton gradient.
The gradient is used to synthesize ATP.
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Acetyl-CoA is formed from the breakdown
of glycogen, fats, and many amino acids.
Oxidation of Acetyl-groups via the citric
acid cycle includes 4 steps in which
electrons are abstracted.
Electrons carried by NADH and FADH2 are
funneled into the electron transport chain
reducing O2 to H2O and producing ATP in the
process of oxidative phosphorylation
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Acetyl CoA
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PYRUVATE
ACETYL COENZYME-A
 Under aerobic conditions, the pyruvate is transported into
the mitochondria in exchange for OH- by the pyruvate
carrier antiporter.
 In the mitochondrial matrix, pyruvate is oxidatively
decarboxylated by the pyruvate dehydrogenase complex to
form acetyl CoA.
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PYRUVATE DEHYDROGENASE COMPLEX
 Pyruvate dehydrogenase is a member of a family of
giant homologous complexes with molecular masses
ranging from 4 -10 million daltons.
 The elaborate structure
of the members of this
family allows groups to
travel from one active
site to another.
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PYRUVATE DEHYDROGENASE COMPLEX REQUIRES 5
COENZYMES
• Catalytic cofactors:
– Thiamine pyrophosphate (TPP)
– Lipoic acid
– FAD serve as catalytic cofactors
• Stoichiometric cofactor:
– CoA
– NAD+
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PYRUVATE DEHYDROGENASE COMPLEX IS COMPOSED
OF 3 ENZYMES
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The mechanism of the pyruvate dehydrogenase
reaction
• Three steps:
– Decarboxylation (Pyruvate dehydrogenase E1).
– Oxidation (Pyruvate dehydrogenase E1)
– Transfer of the resultant acetyl group to CoA
(Dihydrolipoyl transacetylase E2 & Dihydrolipoyl
dehydrogenase E3).
• The 3 must be coupled to preserve the free energy
from the decarboxylation and use it for the formation
of NADH and acetyl-CoA.
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Regeneration of the oxidized form
of lipoamide by E3
summary
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The Pyruvate Dehydrogenase structure
a) Dihydrolipoyl transacetylase E2 (8 catalytic triamers).
b) Pyruvate dehydrogenase E1 (a2 b2 tetramer = 24 cpies)
c) Dihydrolipoyl dehydrogenase E3 (a b diamer = 12 copies)
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Dihydrolipoyl transacetylase E2
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Flexible Linkages Allow Lipoamide to Move Between
Different Active Sites
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Comments:
• The structural integration of three kinds of enzymes
makes the coordinated catalysis of a complex reaction
possible.
• The proximity of one enzyme to another increases the
overall reaction rate and minimizes side reactions.
• All the intermediates in the oxidative decarboxylation
of pyruvate are tightly bound to the complex and are
readily transferred because of the ability of the lipoyllysine arm of E2 to call on each active site in turn
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1. Oxaloacetate & Acetyl Coenzyme A Citrate
• Condensation of a four-carbon unit, oxaloacetate, and
a two-carbon unit, the acetyl group of acetyl CoA.
• This reaction is catalyzed by citrate synthase.
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• Oxaloacetate first condenses with acetyl CoA to form
citryl CoA, which is then hydrolyzed to citrate and
CoA.
• The hydrolysis of citryl CoA, a high-energy thioester
intermediate, drives the overall reaction far in the
direction of the synthesis of citrate.
– In essence, the hydrolysis of the thioester powers the
synthesis of a new molecule from two precursors.
 Because this reaction initiates the cycle, it is very
important that side reactions be minimized.
 How does citrate synthase prevent wasteful processes
such as the hydrolysis of acetyl CoA?
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BY 2 INDUCED FITS
1.
Oxaloacetate, the first substrate bound to the
enzyme, induces a conformational change (1st
induced fit).

A binding site is created for Acetyl-CoA.
Open form
Closed form
2.
Citroyl-CoA formed on the enzyme surface causing a
conformational change (2nd induced fit). The active
site becomes enclosed

2 crucial His and one Asp residues are brought into
position to cleave the thioester of acetyl-CoA and
form citroyl-CoA.
 The dependence of acetyl-CoA hydrolysis on
the two induced fits insures that it is not
hydrolyzed unless the acetyl group is
condensed with oxaloacetate and not
wastefully.
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2. Citrate
•
•
Isocitrate
The isomerization of citrate is accomplished by
a dehydration step followed by a hydration step.
The enzyme catalyzing both steps is called aconitase
because cis-aconitate is an intermediate.
• A 4Fe-4S iron-sulfur cluster is a component of the
active site of aconitase.
• One of the iron atoms of the cluster is free to bind to
the carboxylate and hydroxyl groups of citrate.
3. Isocitrate
a-Ketoglutarate
• The first of four oxidation-reduction reactions in the citric
acid cycle.
• The oxidative decarboxylation of isocitrate is catalyzed by
isocitrate dehydrogenase.
• The intermediate in this reaction is oxalosuccinate, an
unstable b-ketoacid. While bound to the enzyme, it loses
CO2 to form a-ketoglutarate
4. a-Ketoglutarate
Succinyl Coenzyme A
• The second oxidative decarboxylation reaction,
leading to the formation of succinyl-CoA from aketoglutarate.
• This reaction closely resembles that of pyruvate
a-ketoglutarate dehydrogenase complex:
 The complex is homologous to the pyruvate
dehydrogenase complex.
 The reaction mechanism is entirely analogous.
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5. Succinyl Coenzyme A
Succinate
 Succinyl CoA is an energy-rich thioester compound.
 The cleavage of the thioester bond of succinyl CoA is
coupled to the phosphorylation of GDP or ADP.
 This reaction is catalyzed by succinyl CoA synthase
(succinate thiokinase).
Succinyl-CoA synthase:
• An a2b2 heterodimer.
• The functional unit is one ab pair.
• Its mechanism is a clear example of energy
transformations:
– Energy inherent in the thioester molecule is transformed
into phosphoryl-group transfer potential.
• This is the only step in the citric acid cycle that directly
yields a compound with high phosphoryl transfer
potential through a substrate-level phosphorylation.
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1. Displacement of coenzyme A by orthophosphate, which
generates another energy-rich compound, succinyl
phosphate.
2. A His residue of the a subunit removes the phosphoryl group
with the concomitant generation of succinate and
phosphohistidine.
3. The phosphohistidine residue then swings over to a bound
GDP or ADP.
4. The phosphoryl group is transferred to form GTP or ATP.
1
2
3
4
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6. Succinate
Oxaloacetate
• Reactions of four-carbon compounds constitute the final
stage of the citric acid cycle: the regeneration of
oxaloacetate.
• The reactions constitute a metabolic motif that we will see
again:
– A methylene group (CH2) is converted into a carbonyl group (C
= O) in three steps:
•
an oxidation, a hydration, and a second oxidation reaction
STOICHIOMETRY OF THE CITRIC ACID CYCLE
1. Two carbon atoms enter the cycle in the condensation
of an acetyl unit (from acetyl CoA) with oxaloacetate.
Two carbon atoms leave the cycle in the form of CO2
in the successive decarboxylations catalyzed by:
 isocitrate dehydrogenase
 a-ketoglutarate dehydrogenase.
Interestingly, the results of isotope-labeling studies
revealed that the two carbon atoms that enter each
cycle are not the ones that leave.
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2. 4-pairs of hydrogen atoms leave the cycle in four
oxidation reactions.
 Two molecules of NAD+ are reduced in the oxidative
decarboxylations of isocitrate and a-ketoglutarate
 one molecule of FAD is reduced in the oxidation of
succinate
 one molecule of NAD+ is reduced in the oxidation of
malate.
3. One compound with high phosphoryl transfer
potential, usually GTP, is generated from the cleavage
of the thioester linkage in succinyl CoA.
4. Two molecules of water are consumed:
 one in the synthesis of citrate by the hydrolysis of citryl
CoA
 the other in the hydration of fumarate.
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Summary of 8 steps
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REGULATION OF THE PYRUVATE DEHYDROGENASE
COMPLEX:
IRREVERSABLE STEP
&
A BRANCH POINT
Allosteric regulation
High products level
Covalent modification:
Phosphoryl/ dephosphoryl.
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Allosteric
Regulation
NAD+
NADH H+
CoA
CO2
Acetyl-CoA
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Vasopressin
Covalent
Modification
Insulin
Ca+2
+
+
---
ADP
Pyrovate
NAD+
++
NADH
Acetyl-CoA
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The Citric Acid Cycle Is Controlled at Several
Points
 The primary control points are
the allosteric enzymes:
 isocitrate dehydrogenase
 a-ketoglutarate dehydrogenase.
 The citric acid cycle is regulated
primarily by the concentration
of:
 ATP
 NADH.
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Isocitrate dehydrogenase
 Allosterically stimulated by ADP, which enhances the
enzyme's affinity for substrates.
 mutually cooperative binding of:
 Isocitrate
 NAD+
 Mg2+
 ADP.
 NADH inhibits iso-citrate dehydrogenase by directly
displacing NAD+.
 ATP too, is inhibitory.
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a-ketoglutarate dehydrogenase
 Some aspects of this enzyme's control are like those of
the pyruvate dehydrogenase complex.
 inhibited by the products of the reaction that it
catalyzes :
 succinyl CoA
 NADH,.
 high energy charge. The rate of the cycle is reduced
when the cell has a high level of ATP.
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The Citric Acid Cycle Is a Source of Biosynthetic
Precursors
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The citric acid cycle intermediates must be
replenished if consumed in biosyntheses
 An anaplerotic reaction:
 A reaction that leads to the net synthesis, or
replenishment, of pathway components.
 Because the citric acid cycle is a cycle, it can be
replenished by the generation of any of the
intermediates.
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How is oxaloacetate replenished?
 Mammals lack the enzymes for the net conversion of acetyl
CoA into oxaloacetate or any other citric acid cycle
intermediate.
 Oxaloacetate is formed by the carboxylation of pyruvate, in
a reaction catalyzed by the biotin-dependent enzyme
pyruvate carboxylase.
 Acetyl CoA, abundance signifies the need for more
oxaloacetate.
 If the energy charge is high, oxaloacetate is converted into
glucose. If the energy charge is low, oxaloacetate replenishes
the citric acid cycle.
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The glyoxylate
cycle
Allows plants and
some microorganisms
to grow on acetate
because the cycle
bypasses the
decarboxylation steps
of the citric acid cycle.
The enzymes that
permit the conversion
of acetate into
succinate are
isocitrate lyase and
malate synthase.
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