THE CITRIC ACID CYCLE

The final common pathway for the oxidation of fuel molecules amino

acids, fatty acids, and carbohydrates.

In eukaryotes, the reactions of the citric acid

cycle take place inside mitochondria, in contrast with those of glycolysis, which take place in the

cytosol.

An 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 oxidation-reduction reactions that result in the

oxidation of an acetyl group to two molecules of CO2 .

The citric acid cycle is highly efficient:

•Because a limited number of molecules can generate large amounts of NADH and FADH2.

(account for > 95% of energy)•An acetyl group (two-carbon units) is oxidized

to:

.1Two molecules of CO2

.2One molecule of GTP

.3High-energy electrons in the form of NADH and

FADH2 .

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.

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 pfospforylation

Acetyl CoA is the fuel for the citric acid cycle.

PYROVATEACETYL 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 .

PYROVATE 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 structureof the members of thisfamily allows groups totravel from one site to

another.

PYROVATE DEHYDROGENASE COMPLEX REQUIRES 5

COENZYMES•Catalytic cofactors:

–Thiamine pyrophosphate (TPP)–Lipoic acid–FAD serve as catalytic cofactors

•Stoichiometric cofactor:–CoA–NAD+

PYROVATE DEHYDROGENASE COMPLEX IS COMPOSED OF 3

ENZYMESPyruvate dehydrogenase complex of E. coli

EnzymeNumber

of chainsProsthetic

groupReaction catalyzed

Pyruvate

dehydrogenase E1 24TPPOxidative

decarboxylation of pyruvate

Dihydrolipoyl

transacetylase E2 24LipoamideTransfer of the acetyl

group to CoA

Dihydrolipoyl

dehydrogenase E3 12FADRegeneration of the

oxidized form of lipoamide

The mechanism of the pyruvate dehydrogenase reaction

•3 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.

.1Decarboxylation reaction of E1:

•pyruvate combines with TPP–Highly acidic C between N and S; it ionizes to form

carbanion which adds to carbonyl group of pyruvate

•pyruvate is then decarboxylated.

.2Oxidation reaction of E1:

•TPP-hydroxyethyl group is oxidized to form an acetyl group which is concomitantly

transferred to lipoamide.•lipoamide is a derivative of lipoic acid that is

linked to the side chain of a lysine residue by an amide linkage.

•This reaction, also catalyzed by E1, yields acetyllipoamide.

Oxidizing agent

Reduced to disulfhydryl

.3Acetyl group transfer to CoA reaction of E2:

•Dihydrolipoyl transacetylase (E2) catalyzes this reaction.

•The energy-rich thioester bond is preserved as the acetyl group is

transferred to CoA .

Regeneration of the oxidized form of lipoamide by E3:

•In a fourth step, the oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3).

•Two electrons are transferred to an FAD prosthetic group of the enzyme and then to NAD.+

•electron transfer to FAD is unusual, because the common role for FAD is to receive electrons from

NADH.•The electron transfer potential of FAD is altered by its

association with the enzyme and enables it to transfer electrons to NAD .+

summary

a) Dihydrolipoyl transacetylase E2 (8 catalytic triamers).

b) Pyruvate dehydrogenase E1 ( tetramer = 24 cpies)

c) Dihydrolipoyl dehydrogenase E3 ( diamer = 12 copies)

The Pyruvate Dehydrogenase structure

Dihydrolipoyl transacetylase E2

Flexible Linkages Allow Lipoamide to Move Between

Different Active Sites

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 lipoyl-lysine arm of E2 to call

on each active site in turn

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.

•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?

BY TWO INDUCED FITS.1Oxaloacetate, the first substrate bund to the

enzyme, induces a conformational change (1st induced fit).

A binding site is created for Acetyl-CoA.

Open Conformation

Closed Conformation

.2Citroyl-CoA formed on the enzyme surface causing a conformational change (2nd induced

fit). The active site becomes enclosed2 crucial His and one Asp residues are brought into

position to cleave the 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.

CitrateIsocitrate

•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 .

Isocitrate-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 -ketoacid. While bound to the enzyme, it loses

CO2 to form -ketoglutarate

-KetoglutarateSuccinyl Coenzyme A

•The second oxidative decarboxylation reaction, leading to the formation of succinyl-CoA from -

ketoglutarate.•This reaction closely resembels that of pyruvate

-ketoglutarate dehydrogenase complex:

•The complex is homologous to the pyruvate dehydrogenase complex.

•The reaction mechanism is entirely analogous.–The a-ketoglutarate dehydrogenase component

(E2) and transsuccinylase (E1) are different from but homologous to the corresponding enzymes in

the pyruvate dehydrogenase complex

–whereas the dihydrolipoyl dehydrogenase components (E3) of the two complexes are identical

Succinyl Coenzyme ASuccinate

•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 synthetase

(succinate thiokinase) .

Succinyl-CoA synthetase:

n heterodimer.•The functional unit is one 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 .

1. displacement of coenzyme A by orthophosphate, which generates another energy-rich compound, succinyl phosphate.

2. A His residue of the 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

SuccinateOxaloacetate

•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

.1Succinate is oxidized to fumarate by succinate

dehydrogenase •The hydrogen acceptor is FAD rather than NAD+•In succinate dehydrogenase, the isoalloxazine

ring of FAD is covalently attached to a histidine side chain of the enzyme.

•FAD is the hydrogen acceptor in this reaction because the free-energy change

is insufficient to reduce NAD.+

•FAD is nearly always the electron acceptor in oxidations that remove two hydrogen

atoms from a substrate .

Succinate dehydrogenase:•contains three different kinds of iron-sulfur clusters:

.12Fe-2S

.23Fe-4S

.34Fe-4S .•Succinate dehydrogenase consists of two subunits, one

70 kd and the other 27 kd•It differs from other enzymes in the citric acid cycle in

being embedded in the inner mitochondrial membrane.•It is directly associated with the electron-transport chain,

the link between the citric acid cycle and ATP formation.•FADH2 does not dissociate from the enzyme, in contrast

with NADH produced in other oxidation-reduction reactions.

–Two electrons are transferred from FADH2 directly to iron-sulfur clusters of the enzyme.

–The ultimate acceptor of these electrons is molecular oxygen ,

.2Hydration of fumarate to form L-malate:

•Fumarase catalyzes a stereospecific trans

addition of a hydrogen atom and a hydroxyl

group.•The hydroxyl group adds

to only one side of the double bond of fumarate; hence, only the L-isomer

of malate is formed .

.3malate is oxidized to form oxaloacetate:

•This reaction is catalyzed by malate dehydrogenase.

• NAD+ is again the hydrogen acceptor .

STOICHIOMETRY OF THE CITRIC ACID CYCLE

.1Two 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 dehydrogenasea-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.

.24-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-

ketoglutarateone molecule of FAD is reduced in the oxidation of

succinateone molecule of NAD+ is reduced in the oxidation

of malate.

.3One compound with high phosphoryl transfer potential, usually GTP, is generated from the

cleavage of the thioester linkage in succinyl CoA..4Two molecules of water are consumed:

one in the synthesis of citrate by the hydrolysis of citryl CoA

the other in the hydration of fumarate.

CONTROL OF THE CITRIC ACID CYCLE

REGULATION OF THE PYROVATE DEHYDROGENASE COMPLEX:

IRREVERSABLE STEP&

A BRANCH POINT

Allosteric regulationHigh products level

Covalent modification:Phosphoryl/ depfospforyl.

CoA CO2

NAD+ NADH H+NADH H+

Acetyl-CoA

AllostericRegulation

CovalentModification

Acetyl-CoANAD+ NADHPyrovate

-- + +

ADP

-

Ca+2

+ +

InsulinVasopressin

The Citric Acid Cycle Is Controlled at Several Points

•The primary control points are the allosteric enzymes:

–isocitrate dehydrogenase-ketoglutarate

dehydrogenase.

•The citric acid cycle is regulated primarily by the

concentration of:–ATP–NADH.

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.

-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 .

The Citric Acid Cycle Is a Source of Biosynthetic Precursors

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 .

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 .

The glyoxylatecycle

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.