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2 The TCA Cycle: The Fate of Acetyl-CoA

2 The TCA Cycle: The Fate of Acetyl-CoA

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9.2 The TCA Cycle: The Fate of Acetyl-CoA

CO 2-



Co

AS

H



sy

nth

as

e)



3



CH



CH



(aconitase)



CO 2-



H2 O



C



CO 2-



HO CH



CH2



CH 2

CO2 -



CO2 cis-aconitate



citrate



C CO2-



H

(aconitase)



isocitrate

NAD +



ate



O



(ci

tr



C

Co

AS



CH 2



CO2 -



CO2 H 2O



CH 2

HO C CO2 -



211



(isocitrate dehydrogenase)



CO 2-



NADH + H+



C O

CH 2



CO2-



CO 2oxaloacetate



O C

H C CO2 -



NADH + H+

(malate dehydrogenase)



CH2



NAD+



CO2-



CO2 -



oxalosuccinate



HO CH

H 2C



CO2



CO2 L-malate



CO2 -



(fumarase)

H2O



O C

H C



CO2 -



H



CH 2



CH



CO2 -



HC



α-ketoglutarate



-



CO2

fumarate



CoASH

FADH2



NAD+



(succinate dehydrogenase)



(α-ketoglutarate

dehydrogenase)



CO 2



FAD



NADH + H+

CO2 -



CO2 -



CH2



CH2



CH2

CO2 -



+



CH2

CO2 -



CO2



-



GTP



GDP + Pi



CH2



H C H



CH2

CO2 succinate



O C SCoA



CoASH



(succinyl-CoA

synthetase)



CH 2

CO 2succinyl-CoA



Fig. 9.6 The reactions involved in the TCA Cycle. Acetyl-CoA is fed into the cycle, and its two

carbon atoms are converted to oxaloacetate in a single cycle. The carbon atom(s) are traced by

color coding the carbonyl carbon atom (red) of acetyl-CoA which is introduced into the cycle. At

the succinyl-CoA synthetase step the labeled carbonyl carbon atom from acetyl-CoA appears

exclusively in one of the carboxyl groups of succinate; however, because succinate has a twofold

axis of symmetry, it is not possible to distinguish one carboxylate group from the other. Thus, each

carboxylate will appear to contain 50% (blue) of the label from succinyl-CoA



212



9.3



9 The Tricarboxylic Acid Cycle



Energetics of Pyruvate Oxidation



The reoxidation NADH and FADH2 in mitochondria involve redox reactions

that occur with the release of Gibbs free energy which is captured in oxidativephosphorylation as ATP.

When one molecule of NADH is reoxidized to NAD+, 2.5 ATP molecules

are synthesized from ADP ỵ Pi 12 O2 ỵ NADPH ỵ Hỵ ! NADỵ ỵ H2 Oị. In the

case of FADH2 reoxidation, 1.5 ATP molecules are synthesized FADH2 ỵ

1

2 O2 ! FAD ỵ H2 OÞ.

The energetics of specific TCA Cycle reactions are shown in Table 9.1.

Table 9.1 The production of energy (ATP) in mitochondria from pyruvate

Enzyme system

Coenzyme (or nucleotide)

a

NADH

Pyruvate dehydrogenase

Isocitrate dehydrogenase

NADH

a-ketoglutarate dehydrogenase

NADH

Succinate thiokinase

GTP

Succinate dehydrogenase

FADH2

Malate dehydrogenase

NADH

a

The pyruvate dehydrogenase complex is not part of the TCA cycle



ATP equivalence

2.5

2.5

2.5

1

1.5

2.5



Thus, when one molecule of pyruvate is oxidized in the TCA cycle, by the

pyruvate dehydrogenase complex, and its electrons passed on to O2 via oxidative

phosphorylation, 12.5 ATP molecules are produced. Because glucose produces two

pyruvates, 25 ATP molecules are formed in the process of oxidative phosphorylation. In glycolysis, two ATP molecules are produced from glucose. Thus when

glucose is oxidized, a total of 27 ATP molecules are synthesized. Because oxidative

phosphorylation is responsible for 25 of these molecules, 93% of the ATP

molecules generated when glucose is oxidized to CO2 and H2O come from the

pyruvate dehydrogenase complex and the TCA cycle.

The energetics, in terms of redox reactions are illustrated in the following

example: Consider the pyruvate dehydrogenase reaction in which a single NADH

molecule is generated.

The overall reaction for the reoxidation of NADH is:

1



NADH þ Hþ þ O2 ! NADþ þ H2 O;

2



Table 9.2 Reduction

potentials for redox systems



eo ẳ ỵ1:155 V:



Reaction



Reduction potential (V)



ỵ 2Hỵ þ 2eÀ ! H2 O

þ

NAD þHþ þ 2eÀ ! NADH



+0.815

À0.34



1=2O

2



9.4 Stereochemistry of the TCA Cycle



213



The Nernst equation which allows one to convert voltage generated in a redox

reaction to Gibbs free energy is, DG0 ¼ À Â F Â eo , where  is the number

of electrons transferred per mole of reactants, F the Faradays (96.5kJ/Vxmol), and

eo the voltage of the system. Table 9.2 provides the half-cell reduction potentials.

Thus, DG0 ¼ À2  96,500  1.155¼À223kJ/mol. The DG0 of ATP hydrolysis

is À30.5kJ/mol. Because 2.5 ATP molecules are synthesized per NADH oxidized,

the theoretical efficiency of the phosphorylation coupled to oxidation is 34%. In the

living cell, as opposed to the test tube where equilibrium thermodynamics prevail,

the efficiency of oxidation coupled to ATP synthesis is believed to exceed 60%.



9.4



Stereochemistry of the TCA Cycle



Wood and Werkman [2] showed in 1936 that [13C]CO2 was fixed (incorporated)

into biomolecules in bacteria. This was an extremely important discovery because

until then it was believed that only plants could fix CO2. It was later demonstrated

that the isotopic CO2 was incorporated exclusively into one of the carboxyl groups

of a-ketoglutarate. It was concluded from these results that citrate, a symmetrical

molecule, with a onefold axis of symmetry, could not be a member of the Citric

Acid Cycle [3]. Examination of the TCA cycle, if this idea was correct, would

predict that in the aconitase reaction, dehydration could occur either at the top or at

the bottom end of citrate. Thus, there should be a 50–50% distribution of the [13C]

in isocitrate and a-ketoglutarate, and 50% of the synthesized succinate should be

labeled. This conclusion was generally accepted by the biochemical community

until 1948 when Ogston [4] showed that asymmetric attack could occur on

a symmetrical molecule such as citrate. This hypothesis, known as the “Polyaffinity

Theory,” asserts that a symmetrical molecule can be attacked asymmetrically if the

enzyme (itself asymmetrical) makes a three-point attachment to the substrate, i.e.,

this precise three-point attachment is a prerequisite for catalysis. How then does

the Polyaffinity Theory account for asymmetric attack on citrate? Consider the

prochiral carbon center shown in Fig. 9.7.

If the molecule is rotated 109.5 the following interactions between the enzyme

and substrate are obtained.

Thus, making the assumptions described by the Polyaffinity Theory, the enzyme

can discriminate between the two “b” groups on the substrate, i.e., only the

orientation between the substrate and the enzyme described by interaction A, will

allow catalysis to occur. Complexes B and C do not describe a three point attachment between enzyme and substrate, and are therefore not productive.

It should be pointed out that the Polyaffinity Theory is not valid for symmetrical

molecules that have a twofold or greater axis of symmetry, e.g., succinate.



214



9 The Tricarboxylic Acid Cycle

substrate

a d

b



b



b



b



attack site



d



enzyme



binding sites



(BINDING)

a d

b

b



dd

A



a b

b



b



bb



bb



a b

d

b



d

d



d



b

b



b



B



d

C



Fig. 9.7 A prochiral center can be attacked asymmetrically if the enzyme makes a precise threepoint (A) attachment to the substrate. Other binding modes between the enzyme and substrate are

unproductive



9.5

9.5.1



TCA Cycle Enzymes and Their Mechanisms

Citrate Synthase (DG0 ¼ À31.4 kJ/mol)



One of the substrates for citrate synthase, oxaloacetate, has an sp2 prochiral center.

The other substrate, acetyl CoA, adds to the si face of oxaloacetate (Fig. 9.8).



9.5 TCA Cycle Enzymes and Their Mechanisms



215

si face



-



:BE



OH



O

CoAS



C



CoAS



O



H A-E



C



H2 C



CH 3

keto-acetyl-CoA



-O2C



C



CH 2



enol-acetyl-CoA



CO 2oxaloacetate



O



CO 2CH 2

HO



C



CoASH



H2 O



CoAS



-O2C

OH

CH 2 C



C



CH 2



CO 2-



CO2 -



CH 2

CO 2citrate



citryl-CoA



Fig. 9.8 The stereochemistry of the citrate synthase reaction: addition of acetyl-CoA to the si face

of oxaloacetate



9.5.2



Aconitase (DG0 ¼ +5.0 kJ/mol)



Aconatase catalyzes a two step reaction. First, it causes dehydration of citrate to

form cis-aconitate. The second step involves hydration of cis-aconitate to yield

isocitrate. The overall sequence of reactions results in an isomerization of the

symmetrical citrate molecule to isocitrate in which there is no axis of symmetry.

Citrate has a onefold axis of symmetry, and dehydration occurs specifically

at the pro-R arm of the substrate.

In order to maintain the correct stereochemistry of the isomerization reaction,

it is assumed that cis-aconitate dissociates from aconitase, rotates or flips 180 and

then rebinds. The iron–sulfur cluster interacts with the hydroxyl group so as to

weaken the carbon–oxygen bond, thus enhancing the hydroxyl group’s ability to act

as a leaving group. The iron–sulfur cluster and the catalytic base that removes the

HR proton are not believed to move significantly. The function of the iron–sulfur

cluster in aconitase is to bind the hydroxyl group that is first removed, and water

that is added back to the substrate. It is not believed to be involved in a redox-type

reaction in the case of aconitase (Fig. 9.9).



216



9 The Tricarboxylic Acid Cycle

CO2- H



pro-S

arm



CH 2



C



-



O2 C

citrate



Fe



EAH



CO2 -



S



pro-R

arm



C



OH H R CO2



S



Fe



S

Fe



S



HOH



CH 2



HOH

-O



-



2C



:BE

Fe



H



C



Fe



S

Fe



S



Fe

180° flip

CO 2-



H

C



C



CH 2



-O C

2



H

OH



EB:-



HOH



S



Fe



S

Fe



S



CO2 (2R,3S)-isocitrate



S



S



CH 2

C



CO 2cis-aconitate



CO2 -



O2 C



H



C



HOH



-



S

Fe

iron-sulfur cluster



-



C



Fe

S



Fe



CO 2-



H



A-E



cis-aconitate



Fig. 9.9 The stereochemistry and mechanism of action of aconitase. The enzyme makes an

asymmetrical attack on the symmetrical substrate citrate. The proton abstracted from citrate

appears in isocitrate, and the hydroxyl group removed from citrate appears in water



9.5.3



Isocitrate Dehydrogenase (DG0 ¼ À20.9kJ/mol)



Isocitrate dehydrogenase is a typical pyridine-linked anerobic dehydrogenase;

however, one of its reaction products oxalosuccinate is unstable in the presence

of Mn2+, a ubiquitous cation, and spontaneously decarboxylates to form

a-ketoglutarate. Studies with enzyme mutants suggest that the decarboxylation

occurs on the enzyme (Fig. 9.10).



9.5 TCA Cycle Enzymes and Their Mechanisms



217

CO 2-



CO2 -



NAD+



O2 C



C



H



H



C



OH



CH 2

-



O C

O



=



CH 2

-



NADH+H+



H



C



C O

C O-



Mn2+



O



CO2 -



oxalosuccinate-Mn2+ complex



isocitrate



CO2

CO2CO2 CH 2



CH2

H



H C



CH 2



C O



C



C O-



O



CO2 a-ketoglutarate



A-E



Mn 2+



O

carbanion intermediate



Fig. 9.10 The isocitrate dehydrogenase reaction involves both a redox and a decarboxylation

reaction



9.5.4



a-Ketoglutarate Dehydrogenase (DG0 ¼ À32.9kJ/mol)



a-Ketoglutarate dehydrogenase is not a single enzyme but rather a complex similar

to the pyruvate dehydrogenase complex. Like pyruvate dehydrogenase, the basic

unit of the a-ketoglutarate dehydrogenase complex is a trimer, and the coenzymes

are identical to those described for pyruvate dehydrogenase, as is the mechanism.

After completion of the a-ketoglutarate dehydrogenase reaction, acetyl-CoA would

have lost both of its carbon atoms in the form of CO2.



9.5.5



Succinyl-CoA Synthetase (DG0 ¼ À2.1 kJ/mol)



One of the products of the a-ketoglutarate dehydrogenase reaction is succinyl-CoA.

In the presence of GDP and Pi, succinyl-CoA synthetase catalyzes a substrate level

phosphorylation, i.e., GDP is phosphorylated by Pi to form GTP at the expense of

succinyl-CoA, which like acetyl-CoA is a “high energy compound” (Chap. 7).



218



9 The Tricarboxylic Acid Cycle

CoASH



CO 2-



CO2 -



CH 2



CH 2



CH 2



CH 2



O



C O



C O



-



O P



CoAS



OH



OH



N



NH



O P O-



AE



OH



phosphate



succinyl-CoA



E



O



histidyl-E



succinyl phosphate

CO2 O

G



O



O

-



O



O



O



-



O-



NH



O



phosphohistidine-E



O



CH 2 O P O P O P OH

O-



E

N+



HO P



-



GDP



HO OH



O



O: -



CH 2 O P O P

O



G



O



O-



+



CH 2

CH 2

CO2 succinate



E

+



N



NH



histidyl-E

HO OH



GTP



Fig. 9.11 Succinyl-CoA synthetase (succinate thiokinase) is involved in a substrate level phosphorylation. Enzyme-bound phosphohstidine is involved in covalent catalysis



The succinyl-CoA synthetase reaction involves the participation of a phosphohistidine intermediate, an example of covalent catalysis. Investigation of this

enzyme in Boyer’s laboratory in 1964 was the first example of the involvement of

phosphohistidine as an intermediate in an enzyme catalyzed reaction [5] (Fig. 9.11).



9.5.6



Succinate Dehydrogenase (DG0 ¼ + 6.0 kJ/mol)



Succinate dehydrogenase oxidizes the symmetrical molecule succinate to fumarate

by removing a pro-HR from one carbon atom and a pro-HS from the other. The

hydrogens on the product are thus trans. One of the hydrogens leaves succinate as

a proton and the other as a hydride ion. FAD is covalently bound to the enzyme in

yeast through a histidine residue [6]. The reduced coenzyme FADH2 is reoxidized

by the electron transport chain (Fig. 9.12).



9.5 TCA Cycle Enzymes and Their Mechanisms

Fig. 9.12 The mechanism

of the FAD-linked enzyme

succinate dehydrogenase



219



H3 C



R

N



H3 C



N

HS

HR



-O 2C

C



C



HS

succinate



HR



H

N



A-E

O



NH

O

FAD



CO2-



EB:-



-O2 C

C



H



H3 C



R

N



CO 2 -



H3 C



N



H

N



C



H



O

NH



O

H

FADH2



fumarate



Fumarase (DG0 ¼ À3.4 kJ/mol)



9.5.7



The fumarase mechanism of action is not a settled issue at this time. Two proposals

have been advanced that accord with data available in the literature. One involves

a carbocation intermediate and the other a carbanion intermediate. The two mechanisms are illustrated in Fig. 9.13.



-



O 2C



H



H

O



H



C



H



-



H

C



O 2C



CO 2-



H



fumarate



C

OH



H



OH



C



C

-O2C H



CO2 -



C

H CO2



(S)-malate



H A-E

carbanion intermediate



EB: -



H

O

H



-



O2 C

H



C



-



O2 C



C

CO2 -



H

C



C

CO 2-



H



H



:BE



H



-



O 2C



-



H



C



C

H CO2 -



fumarate

E-A



H



carbocation intermediate



Fig. 9.13 Shown here are two proposals that have been advance as mechanisms for fumarase.

The upper mechanism represents a hydration reaction involving a carbanion intermediate, whereas

the intermediate in the lower mechanism is a carbocation



220



9.5.8



9 The Tricarboxylic Acid Cycle



Malate Dehydrogenase (DG0 ¼ +29.6 kJ/mol)



Malate dehydrogenase is the last enzyme in the TCA cycle. In this step malate is

oxidized to oxaloacetate at the expense of NAD+. The NADH produced in all of the

TCA cycle reactions as well as FADH2 generated at the succinate dehydrogenase

step are reoxidized by the ETS, which is on the surface of the inner mitochondrial

membrane, and thus in close proximity to the enzymes and products of the TCA

cycle. With the formation of oxaloacetate from malate, the TCA cycle is completed

and poised to accept a new molecule of acetyl-CoA for oxidation.



9.6



Regulation of Acetyl-CoA Oxidation



Control of acetyl-CoA degradation occurs at the pyruvate dehydrogenase and TCA

cycle levels.



9.6.1



Pyruvate Dehydrogenase Regulation



The pyruvate dehydrogenase complex can be inhibited by covalent modification

(phosphorylation of a serine residue on E1), and by products NADH and acetyl-CoA.

The enzyme responsible for phosphorylation of E1, pyruvate dehydrogenase kinase,

is activated by high levels of ATP, acetyl-CoA, and NADH and inhibited by pyruvate,

and ADP. The fundamental role of the pyruvate dehydrogenase complex and the

TCA cycle is to produce ATP from NADH and FADH2. When ATP, NADH, and

acetyl-CoA levels are high, pyruvate dehydrogenase kinase activity is activated and

the flux through the pyruvate dehydrogenase complex is inhibited. Pyruvate dehydrogenase phosphatase, the enzyme that dephosphorylates phosphorylated E1, is

itself activated by the hormone insulin [7] and Mg2+. The end result of phosphatase activation is the enhancement of acetyl-CoA degradation (Fig. 9.14).



9.6 Regulation of Acetyl-CoA Oxidation



221



ATP, Acetyl-CoA,

NADH

S S



ADP



(+)



pyruvate dehydrogenase

kinase

FAD



TPP

E1



E2



active



S S



(-)



E1



E3

ATP

Pi



FAD



TPP

E2



E3



ADP

H2O



inactive

P



pyruvate dehydrogenase

phosphatase



(+)

insulin, Mg2+



Fig. 9.14 Regulation of pyruvate dehydrogenase by phosphorylation and dephosphorylation of

subunit E1



9.6.2



TCA Cycle Regulation



The metabolic flux through the TCA cycle is controlled by factors such as the

availability of acetyl-CoA, the rates of reoxidation of NADH and FADH2, and the

level of TCA cycle intermediates. It has been suggested that regulation of the TCA

cycle depends upon those enzymes, such as citrate synthase, which are highly

exergonic and catalyze “irreversible steps” in the cycle. As was shown earlier

(Chap. 7), it is the mass action ratio and not exclusively the equilibrium constant

of enzymes that determines whether they are potential regulatory steps in a metabolic pathway.

In vitro studies with various TCA cycle enzymes indicate that citrate is a competitive inhibitor of oxaloacetate in the citrate synthase reaction and that succinyl-CoA

competes with acetyl-CoA in the citrate synthase reaction. In addition, Ca2+ is

an activator of both pyruvate and a-ketogluterate dehydrogenase reactions.

The regulation of the TCA Cycle is summarized in Fig. 9.15.



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