Citric Acid Cycle: Understanding the Role of Citrate Synthase and Succinyl CoA Synthetase , Study notes of Biochemistry

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Biochemistry: A Short Course

First Edition

Biochemistry: A Short Course

First Edition

Tymoczko • Berg • Stryer

© 2010 W. H. Freeman and Company

CHAPTER 18

Harvesting Electrons from the Cycle

Citric Acid Cycle Generates Reductive

Equivalents Utilized in Respiration

Citrate Synthase Forms

Citrate Synthase Forms

Citrate from Oxaloacetate

Citrate from Oxaloacetate

and Acetyl Coenzyme A

and Acetyl Coenzyme A

The citric acid cycle

The citric acid cycle

begins with the

begins with the

condensation of a four-

condensation of a four-

carbon unit,

carbon unit,

oxaloacetate, and a two-

oxaloacetate, and a two-

carbon unit, the acetyl

carbon unit, the acetyl

group of acetyl CoA.

group of acetyl CoA.

catalyzed by

catalyzed by

citrate

citrate

synthase.

synthase.

Oxaloacetate + acetyl CoA

Oxaloacetate + acetyl CoA

citryl CoA

citryl CoA





citrate

citrate

and CoA.

and CoA.

Mammalian

Mammalian

citrate synthase

citrate synthase

is a

is a

dimer of

dimer of

identical 49-kd subunits

identical 49-kd subunits

. Each active site

. Each active site

is located in a cleft between the large and

is located in a cleft between the large and

small domains of a subunit, adjacent to the

small domains of a subunit, adjacent to the

subunit interface. Citrate synthase exhibits

subunit interface. Citrate synthase exhibits

sequential, ordered kinetics:

sequential, ordered kinetics:

oxaloacetate

oxaloacetate

binds first, followed by acetyl CoA

binds first, followed by acetyl CoA

. The

. The

reason for the ordered binding is that

reason for the ordered binding is that

oxaloacetate induces a major structural

oxaloacetate induces a major structural

rearrangement leading to the creation of a

rearrangement leading to the creation of a

binding site for acetyl CoA.

binding site for acetyl CoA.

Mechanism of Synthesis of Citryl CoA

Mechanism of Synthesis of Citryl CoA

by

by Citrate Synthase.

Citrate Synthase.

The condensation

The condensation

of oxaloacetate and acetyl CoA

of oxaloacetate and acetyl CoA

proceeds through an

proceeds through an enol

enol

intermediate

intermediate

. The subsequent . The subsequent

hydrolysis of citryl CoA yields

hydrolysis of citryl CoA yields

citrate and CoA.

citrate and CoA.

Citrate Is Isomerized into Isocitrate

Citrate Is Isomerized into Isocitrate

The tertiary hydroxyl group is not properly

The tertiary hydroxyl group is not properly

located in the citrate molecule for the

located in the citrate molecule for the

oxidative decarboxylations

oxidative decarboxylations

that follow. Thus,

that follow. Thus,

citrate is

citrate is

isomerized into isocitrate

isomerized into isocitrate

to

to

enable the six-carbon unit to undergo

enable the six-carbon unit to undergo

oxidative decarboxylation.

oxidative decarboxylation.

The isomerization of citrate is accomplished

The isomerization of citrate is accomplished

by a

by a

dehydration

dehydration

step followed by a

step followed by a

hydration

hydration

step.

step.

The enzyme catalyzing both steps is called

The enzyme catalyzing both steps is called

aconitase

aconitase

because cis

because cis

-aconitate

-aconitate

is an

is an

intermediate

intermediate

Aconitase

Aconitase

is an

is an

iron-sulfur protein

iron-sulfur protein

, or

, or

nonheme

nonheme

iron protein.

iron protein.

four iron atoms

four iron atoms

The four iron atoms are complexed to four

The four iron atoms are complexed to four

inorganic sulfides and three cysteine sulfur

inorganic sulfides and three cysteine sulfur

atoms

atoms

leaving one iron atom available to bind

leaving one iron atom available to bind

citrate

citrate

and then isocitrate through their

and then isocitrate through their

carboxylate and hydroxyl groups.

carboxylate and hydroxyl groups.

Binding of

Binding of

Citrate to the

Citrate to the

Iron-Sulfur

Iron-Sulfur

Complex of

Complex of

Aconitase

Aconitase

. A

. A

4Fe-4S iron-

4Fe-4S iron-

sulfur cluster

sulfur cluster

is a component

is a component

of the active

of the active

site of

site of

aconitase. One

aconitase. One

of the iron

of the iron

Is

Is

ocitrate Is Oxidized and

ocitrate Is Oxidized and

Decarboxylated Decarboxylated toto α-α-

Ketoglutarate Ketoglutarate

now now toto thethe firstfirst ofof fourfour

oxidation-reduction oxidation-reduction reactionsreactions

in in thethe citriccitric acidacid cycle.cycle. TheThe

oxidative oxidative decarboxylationdecarboxylation ofof

isocitrate isocitrate isis catalyzedcatalyzed byby

isocitrate isocitrate dehydrogenasedehydrogenase.. TheThe

intermediate intermediate inin thisthis reactionreaction

is

is oxalosuccinate,

oxalosuccinate, an

an

unstable β-ketoacid. While

unstable β-ketoacid. While

bound to the enzyme, it

bound to the enzyme, it loses

loses

CO

CO

22

to form

to form α-ketoglutarate.

α-ketoglutarate.

This oxidation generates the

This oxidation generates the

first high-transfer-potential

first high-transfer-potential

electron carrier NADH in the

electron carrier NADH in the

cycle.

cycle.

Succinyl Coenzyme A

Is Formed by the

Oxidative

Decarboxylation of

α-Ketoglutarate

The oxidative

The oxidative

decarboxylation of α-

decarboxylation of α-

ketoglutarate closely

ketoglutarate closely

resembles that of

resembles that of

pyruvate, also an α-

pyruvate, also an α-

ketoacid.

ketoacid.

cleavage of the thioester bond

cleavage of the thioester bond

powers the synthesis of the

powers the synthesis of the

six-carbon citrate

six-carbon citrate from the

from the

four-carbon oxaloacetate

four-carbon oxaloacetate and

and

the

the two-carbon fragment.

two-carbon fragment. The

The

cleavage of the thioester bond

cleavage of the thioester bond

of succinyl CoA is coupled to

of succinyl CoA is coupled to

the phosphorylation of a

the phosphorylation of a

purine nucleoside diphosphate,

purine nucleoside diphosphate,

usually GDP.

usually GDP.

This reaction is catalyzed by

This reaction is catalyzed by

succinyl CoA synthetase

succinyl CoA synthetase

(succinate thiokinase). Some

(succinate thiokinase). Some

mammalian

mammalian succinyl CoA

succinyl CoA

synthetases

synthetases are specific

are specific

for

for GDP and others for ADP

GDP and others for ADP .

The

The E. coli

E. coli enzyme uses either

enzyme uses either

GDP or GDP as the

GDP or GDP as the phosphoryl-

phosphoryl-

group acceptor

group acceptor .

Reaction Mechanism of

Succinyl CoA

Synthetase. The

formation of GTP at

the expense of

succinyl CoA is an

example of substrate-

level

phosphorylation. The

reaction proceeds

through a

phosphorylated enzyme

intermediate.

Oxaloacetate Is Regenerated by the Oxidation of

Oxaloacetate Is Regenerated by the Oxidation of

Succinate

Succinate

Reactions of four-carbon compounds constitute the final

Reactions of four-carbon compounds constitute the final

stage of the citric acid cycle: the regeneration of

stage of the citric acid cycle: the regeneration of

oxaloacetate. A methylene group (CH

oxaloacetate. A methylene group (CH

22

) is converted into a

) is converted into a

carbonyl group (C = O) in three steps: an oxidation, a

carbonyl group (C = O) in three steps: an oxidation, a

hydration, and a second oxidation reaction. Not only is

hydration, and a second oxidation reaction. Not only is

oxaloacetate thereby regenerated for another round of

oxaloacetate thereby regenerated for another round of

the cycle, but also more energy is extracted in the form

the cycle, but also more energy is extracted in the form

of FADH

of FADH

2

2

and NADH.

and NADH.

Succinate is oxidized to fumarate by

Succinate is oxidized to fumarate by succinate

succinate

dehydrogenase

dehydrogenase .

The hydrogen acceptor is FAD. In

The hydrogen acceptor is FAD. In

succinate dehydrogenase, the isoalloxazine ring of FAD

succinate dehydrogenase, the isoalloxazine ring of FAD

is covalently attached to a histidine side chain of the

is covalently attached to a histidine side chain of the

enzyme (denoted E-FAD).

enzyme (denoted E-FAD).

FAD is the hydrogen acceptor

FAD is the hydrogen acceptor

in this reaction because the

in this reaction because the

free-energy change is

free-energy change is

insufficient to reduce NAD

insufficient to reduce NAD

++

FAD is nearly always the

FAD is nearly always the

electron acceptor in

electron acceptor in

oxidations that remove two

oxidations that remove two

hydrogen

hydrogen atoms

atoms from a

from a

substrate.

substrate.

Succinate dehydrogenase

Succinate dehydrogenase , like

, like

aconitase

aconitase , is an

, is an iron-sulfur

iron-sulfur

protein

protein

. Indeed, succinate . Indeed, succinate

dehydrogenase contains three

dehydrogenase contains three

different kinds of

different kinds of iron-sulfur

iron-sulfur

clusters, 2Fe-2S

clusters, 2Fe-2S (two iron

(two iron

atoms bonded to two inorganic

atoms bonded to two inorganic

sulfides), 3Fe-4S, and 4Fe-4S.

sulfides), 3Fe-4S, and 4Fe-4S.

Succinate dehydrogenase—which

Succinate dehydrogenase—which

consists of two subunits, one

consists of two subunits, one

70 kd and the other 27

70 kd and the other 27 kd—

kd—

differs from other enzymes in

differs from other enzymes in

the citric acid cycle in being

the citric acid cycle in being