Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

FIGURE 20.10

20.6 ● Isocitrate Dehydrogenase—The First Oxidation in the Cycle

651

20.6 ● Isocitrate Dehydrogenase—The First

Oxidation in the Cycle

In the next step of the TCA cycle, isocitrate is oxidatively decarboxylated to yield -ketoglutarate, with concomitant reduction of NAD to NADH in the isocitrate dehydrogenase reaction (Figure 20.10). The reaction has a net G° of 8.4 kJ/mol, and it is sufficiently exergonic to pull the aconitase reaction forward. This two-step reaction involves (1) oxidation of the C-2 alcohol of isocitrate to form oxalosuccinate, followed by (2) a -decarboxylation reaction that expels the central carboxyl group as CO2, leaving the product -ketoglu- tarate. Oxalosuccinate, the -keto acid produced by the initial dehydrogenation reaction, is unstable and thus is readily decarboxylated.

Isocitrate Dehydrogenase Links the TCA

Cycle and Electron Transport

Isocitrate dehydrogenase provides the first connection between the TCA cycle and the electron transport pathway and oxidative phosphorylation, via its production of NADH. As a connecting point between two metabolic pathways, isocitrate dehydrogenase is a regulated reaction. NADH and ATP are allosteric inhibitors, whereas ADP acts as an allosteric activator, lowering the Km for isocitrate by a factor of 10. The enzyme is virtually inactive in the absence of ADP. Also, the product, -ketoglutarate, is a crucial -keto acid for aminotransferase reactions (see Chapters 14 and 27), connecting the TCA cycle (that is, carbon metabolism) with nitrogen metabolism.

(a)	H2C								COO–
H			C						COO–
H									COO–
			C

OH

NAD+

Isocitrate dehydrogenase

NADH

+ H+

H2C

COO–

CO2

O

H2C

COO–

H

C

C

O–

H2C

H+

C

C

COO–

COO–

O

O

Oxalosuccinate

α -Ketoglutarate

(b)

● (a) The isocitrate dehydrogenase reaction. (b) The active site of isocitrate dehydrogenase. Isocitrate is shown in green, NADP is shown in gold, with Ca2 in red.

652 Chapter 20 ● The Tricarboxylic Acid Cycle

20.7 ● -Ketoglutarate Dehydrogenase—

A Second Decarboxylation

A second oxidative decarboxylation occurs in the -ketoglutarate dehydrogenase reaction (Figure 20.11). Like the pyruvate dehydrogenase complex,-ketoglutarate dehydrogenase is a multienzyme complex—consisting of

-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase—that employs five different coenzymes (Table 20.2). The dihydrolipoyl dehydrogenase in this reaction is identical to that in the pyruvate dehydrogenase reaction. The mechanism is analogous to that of pyruvate dehydrogenase, and the free energy changes for these reactions are 29 to 33.5 kJ/mol. As with the pyruvate dehydrogenase reaction, this reaction produces NADH and a thioester product—in this case, succinyl-CoA. Succinyl-CoA and NADH products are energy-rich species that are important sources of metabolic energy in subsequent cellular processes.

20.8 ● Succinyl-CoA Synthetase—A Substrate-

Level Phosphorylation

The NADH produced in the foregoing steps can be routed through the electron transport pathway to make high-energy phosphates via oxidative phosphorylation. However, succinyl-CoA is itself a high-energy intermediate and is utilized in the next step of the TCA cycle to drive the phosphorylation of GDP to GTP (in mammals) or ADP to ATP (in plants and bacteria). The reaction (Figure 20.12) is catalyzed by succinyl-CoA synthetase, sometimes called succinate thiokinase. The free energies of hydrolysis of succinyl-CoA and GTP or ATP are similar, and the net reaction has a G° of 3.3 kJ/mol. Succinyl-CoA synthetase provides another example of a substrate-level phosphorylation (Chapter 19), in which a substrate, rather than an electron transport chain or proton gradient, provides the energy for phosphorylation. It is the only such reaction in the TCA cycle. The GTP produced by mammals in this reaction can exchange its terminal phosphoryl group with ADP via the nucleoside diphosphate kinase reaction:

Nucleoside diphosphate

kinase

3888888888888888888884

GTP ADP 88888888888888888888 ATP GDP

NADH

NAD+

CoA

+ H+

CO2

H2C

COO–

H2C

COO–

H2C

H2C

C

COO–

α -Ketoglutarate

C

SCoA

O

dehydrogenase

O

α -Ketoglutarate

Succinyl-CoA

FIGURE 20.11 ● The -ketoglutarate dehydrogenase reaction.

20.8 ● Succinyl-CoA Synthetase—A Substrate-Level Phosohorylation

653

Table 20.2

Composition of the -Ketoglutarate Dehydrogenase Complex from E. coli

					Number of
		Enzyme	Number of	Subunit	Subunits
Enzyme	Coenzyme	Mr	Subunits	Mr	per Complex

-Ketoglutarate dehydrogenase	Thiamine pyrophosphate	192,000	2	96,000	24
Dihydrolipoyl transsuccinylase	Lipoic acid, CoASH	1,700,000	24	70,000	24
Dihydrolipoyl dehydrogenase	FAD, NAD	112,000	2	56,000	12
		E	+ Succinyl	CoA

The Mechanism of Succinyl-CoA Synthetase

The mechanism of succinyl-CoA synthetase is postulated to involve displacement of CoA by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active-site histidine (making a phosphohistidine intermediate) and release of succinate. The phosphoryl moiety is then transferred to GDP to form GTP (Figure 20.13). This sequence of steps “preserves” the energy of the thioester bond of succinyl-CoA in a series of high-energy intermediates that lead to a molecule of ATP:

Thioester 88n [succinyl-P] 88n [phosphohistidine] 88n GTP 88n ATP

The First Five Steps of the TCA Cycle Produce

NADH, CO2, GTP (ATP), and Succinate

This is a good point to pause in our trip through the TCA cycle and see what has happened. A two-carbon acetyl group has been introduced as acetyl-CoA and linked to oxaloacetate, and two CO2 molecules have been liberated. The cycle has produced two molecules of NADH and one of GTP or ATP, and has left a molecule of succinate.

The TCA cycle can now be completed by converting succinate to oxaloacetate. This latter process represents a net oxidation. The TCA cycle breaks it down into (consecutively) an oxidation step, a hydration reaction, and a second oxidation step. The oxidation steps are accompanied by the reduction of an [FAD] and an NAD . The reduced coenzymes, [FADH2] and NADH, subsequently provide reducing power in the electron transport chain. (We see in Chapter 24 that virtually the same chemical strategy is used in -oxidation of fatty acids.)

H2C

COO –

H2C

C

SCoA

O

O

–O

P

OH

O–

CoASH

COO –

H2C

H2C

O

O

O–

C

P

O

O–

H2C

COO –

COO –

H2C

Succinate

O

+

N

NH

O P

O–

NH

N

NH

N

H2C				COO–		GDP + P GTP + CoA
H2C								H2C			COO–
											COO–
C				SCoA		Succinyl-CoA		H2C
O						synthetase
Succinyl-CoA								Succinate

FIGURE 20.12 ● The succinyl-CoA synthetase reaction.

GDP

GTP

N	NH

FIGURE 20.13 ● The mechanism of the suc- cinyl-CoA synthetase reaction.

FIGURE 20.15

Histidine

FIGURE 20.14

654 Chapter 20 ● The Tricarboxylic Acid Cycle

					FAD	FADH2
	COO–							H		COO–
		CH2							C
					Succinate
		CH2			dehydrogenase			–OOC	C
			COO–							H
Succinate					FAD			Fumarate
						FADH2

● The succinate dehydrogenase reaction. Oxidation of succinate occurs with reduction of [FAD]. Reoxidation of [FADH2] transfers electrons to coenzyme Q.

C– 8a	R
HN N CH2	N N O
H3C	NH
	N
E	O
	C– 6

FAD

● The covalent bond between FAD and succinate dehydrogenase involves the C-8a methylene group of FAD and the N-3 of a histidine residue on the enzyme.

Cys		Cys
S	S	S
	Fe	Fe
S	S	S
Cys		Cys

FIGURE 20.16 ● The Fe2S2 cluster of succinate dehydrogenase.

20.9 ● Succinate Dehydrogenase—An Oxidation Involving FAD

The oxidation of succinate to fumarate (Figure 20.14) is carried out by succinate dehydrogenase, a membrane-bound enzyme that is actually part of the electron transport chain. As will be seen in Chapter 21, succinate dehydrogenase is part of the succinate–coenzyme Q reductase of the electron transport chain. In contrast with all of the other enzymes of the TCA cycle, which are soluble proteins found in the mitochondrial matrix, succinate dehydrogenase is an integral membrane protein tightly associated with the inner mitochondrial membrane. Succinate oxidation involves removal of H atoms across a COC bond, rather than a COO or CON bond, and produces the trans- unsaturated fumarate. This reaction (the oxidation of an alkane to an alkene) is not sufficiently exergonic to reduce NAD , but it does yield enough energy to reduce [FAD]. (By contrast, oxidations of alcohols to ketones or aldehydes are more energetically favorable and provide sufficient energy to reduce NAD .) This important point is illustrated and clarified in an example in Chapter 21.

Succinate dehydrogenase is a dimeric protein, with subunits of molecular masses 70 kD and 27 kD (see Table 20.1). FAD is covalently bound to the larger subunit; the bond involves a methylene group of C-8a of FAD and N-3 of a histidine on the protein (Figure 20.15). Succinate dehydrogenase also contains three different iron–sulfur clusters (Figure 20.16). Viewed from either end of the succinate molecule, the reaction involves dehydrogenation , to a carbonyl (actually, a carboxyl) group. The dehydrogenation is stereospecific (Figure 20.14), with the pro-S hydrogen removed from one carbon atom and the pro-R hydrogen removed from the other. The electrons captured by [FAD] in this reaction are passed directly into the iron–sulfur clusters of the enzyme and on to coenzyme Q(UQ). The covalently bound FAD is first reduced to [FADH2] and then reoxidized to form [FAD] and the reduced form of coenzyme Q, UQH2. Electrons captured by UQH2 then flow through the rest of the electron transport chain in a series of events that is discussed in detail in Chapter 21.

Note that flavin coenzymes can carry out either one-electron or twoelectron transfers. The succinate dehydrogenase reaction represents a net two-electron reduction of FAD.

20.10 ● Fumarase Catalyzes Trans-Hydration of Fumarate

Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate (Figure 20.17). The reaction involves trans-addition of the elements of water across the double bond. Recall that aconitase carries out a similar reaction,

H

COO–

H

O

OH

2

COO–

C

H

C

COO–

Fumarase

C

H2C

–OOC H

Fumarate

Malate

FIGURE 20.17 ● The fumarase reaction.

20.11 ● Malate Dehydrogenase—Completing the Cycle

655

Carbonium ion mechanism

COO–

COO–

HO–

COO–

+C

H

HO

C

H

C

H

H

C

H

H

C

H

CH2

COO–

B

COO–

COO–

Fumarate

E

Carbonium ion

L-Malate

Carbanion mechanism

COO–

HO–

COO–

COO–

C

H

HO

C

H

HO

C

H

–

H

C

H

C

CH2

H

H

COO–

B

COO– B

COO–

Fumarate

E

Carbanion

E

L-Malate

FIGURE 20.18 ● Two possible mechanisms for the fumarase reaction.

and that trans-addition of OH and OOH occurs across the double bond of cis-aconitate. Though the exact mechanism is uncertain, it may involve protonation of the double bond to form an intermediate carbonium ion (Figure 20.18) or possibly attack by water or OH anion to produce a carbanion, followed by protonation.

20.11 ● Malate Dehydrogenase—Completing the Cycle

In the last step of the TCA cycle, L-malate is oxidized to oxaloacetate by malate dehydrogenase (Figure 20.19). This reaction is very endergonic, with a G° of 30 kJ/mol. Consequently, the concentration of oxaloacetate in the mitochondrial matrix is usually quite low (see the following example). The reaction, however, is pulled forward by the favorable citrate synthase reaction. Oxidation of malate is coupled to reduction of yet another molecule of NAD , the third one of the cycle. Counting the [FAD] reduced by succinate dehydrogenase, this makes the fourth coenzyme reduced through oxidation of a single acetate unit.

NADH

NAD+

+ H+

OH

O

COO–

COO–

H

C

Malate

C

COO–

dehydrogenase

COO–

H2C

H2C

NAD+

NADH

L-Malate

Oxaloacetate

+ H+

FIGURE 20.19 ● The malate dehydrogenase reaction.

656 Chapter 20 ● The Tricarboxylic Acid Cycle

A D E E P E R L O O K

Steric Preferences in NAD -Dependent Dehydrogenases

As noted in Chapter 18, the enzymes that require nicotinamide coenzymes are stereospecific and transfer hydride to either the pro-R or the pro-S positions selectively. The table (facing page) lists the preferences of several dehydrogenases.

What accounts for this stereospecificity? It arises from the fact that the enzymes (and especially the active sites of enzymes) are inherently asymmetric structures. The nicotinamide coenzyme (and the substrate) fit the active site in only one way. Malate

dehydrogenase, the citric acid cycle enzyme, transfers hydride to the HR position of NADH, but glyceraldehyde-3-P dehydrogenase in the glycolytic pathway transfers hydride to the HS position, as shown in the accompanying table. Dehydrogenases are stereospecific with respect to the substrates as well. Note that alcohol dehydrogenase removes hydrogen from the pro-R position of ethanol and transfers it to the pro-R position of NADH.

OH

H C COO– +

H2C COO–

L-Malate

H O

C

H C OH +

H2C OPO=3

Glyceraldehyde-

3-phosphate

HS

HR C OH +

CH3

Ethanol

H

O

O

O

H

HS

C

NH2

COO–

R

C NH2

C

+

+ H+

Malate

COO–

N

+

H2C

N

dehydrogenase

Oxaloacetate

R

R

NAD+

NADH

H

O

HR H

O

O

=

OPO3

S

C

NH2

C

+

C

NH2

+ H+

OH

N

+

Glyceraldehyde-

H

C

N

3-phosphate

OPO=3

dehydrogenase

H2C

R

R

1,3-Bisphospho-

NADH

NAD+

glycerate

H

O

O

H

HS

C

NH2

O

H

R

+

C

+ H+

C

NH2

N

+

Alcohol

CH3

N

dehydrogenase

R

Acetaldehyde

R

NAD+

NADH

NAD(P) -dependent enzymes are stereospecific. Malate dehydrogenase, for example, transfers a hydride to the pro-R position of NADH, whereas glyceraldehyde-3-phosphate dehydrogenase transfers a hydride to the pro-S position of the nicotinamide. Alcohol dehydrogenase removes a hydride from the pro-R position of ethanol and transfers it to the pro -R position of NADH.

20.11 ● Malate Dehydrogenase—Completing the Cycle

657

Steric Specificity for NAD of Various Pyridine Nucleotide-Linked Enzymes

			Steric
Dehydrogenase	Source		Specificity
Alcohol (with ethanol)	Yeast, Pseudomonas, liver,
	wheat germ
Alcohol (with isopropyl alcohol)	Yeast
Acetaldehyde	Liver
L-Lactate	Heart muscle, Lactobacillus		HR
L-Malate	Pig heart, wheat germ
D-Glycerate	Spinach								HR
				R		N
Dihydroorotate	Zymobacterium oroticum								HS
-Glycerophosphate	Muscle							C	O
Glyceraldehyde-3-P	Yeast, muscle
L-Glutamate	Liver							NH2
D-Glucose	Liver
-Hydroxysteroid	Pseudomonas
			HS
NADH cytochrome c reductase	Rat liver mitochondria,
	pig heart
NADPH transhydrogenase	Pseudomonas
NADH diaphorase	Pig heart
L- -Hydroxybutyryl-CoA	Heart muscle

		O				H
						O H
	CH3	C
			H	+	CH3 C
		H
						H
C	C	H			C	C
R N		C			R N	C H
C	C		H+		C	C
	C					C
	NH2	O			NH2	O

The stereospecificity of hydride transfer in dehydrogenases is a consequence of the asymmetric nature of the active site.

Adapted from Kaplan, N. O., 1960. In The Enzymes, vol. 3, p. 115, edited by Boyer, Lardy, and Myrbäck. New

York: Academic Press.

FIGURE 20.20

658 Chapter 20 ● The Tricarboxylic Acid Cycle

EXAMPLE

A typical intramitochondrial concentration of malate is 0.22 mM. If the [NAD ]/[NADH] ratio in mitochondria is 20 and if the malate dehydrogenase reaction is at equilibrium, calculate the intramitochondrial concentration of oxaloacetate at 25°C.

SOLUTION

For the malate dehydrogenase reaction,

Malate NAD 34 oxaloacetate NADH H with the value of G° being 30 kJ/mol. Then

G° RT ln K eq

[1]x(8.314 J mol.K) (298) ln [20][2.2 10 4]

30,000 J mol ln (x 4.4 10 3) 2478 J mol

12.1 ln (x 4.4 10 3)

x (5.6 10 6)(4.4 10 3) x [oxaloacetate] 0.024 M

Malate dehydrogenase is structurally and functionally similar to other dehydrogenases, notably lactate dehydrogenase (Figure 20.20). Both consist of alternating -sheet and -helical segments. Binding of NAD causes a conformational change in the 20-residue segment that connects the D and E strands of the -sheet. The change is triggered by an interaction between the adenosine phosphate moiety of NAD and an arginine residue in this loop region. Such a conformational change is consistent with an ordered single-displacement mechanism for NAD -dependent dehydrogenases (Chapter 14).

(b)

● (a) The structure of malate dehydrogenase. (b) The active site of malate dehydrogenase. Malate is shown in red; NAD is blue.

20.12 ● A Summary of the Cycle

659

20.12 ● A Summary of the Cycle

The net reaction accomplished by the TCA cycle, as follows, shows two molecules of CO2, one ATP, and four reduced coenzymes produced per acetate group oxidized. The cycle is exergonic, with a net G° for one pass around the cycle of approximately 40 kJ/mol. Table 20.1 compares the G° values for the individual reactions with the overall G° for the net reaction.

Acetyl-CoA 3 NAD [FAD] ADP Pi 2 H2O 34

2 CO2 3 NADH 3 H [FADH2] ATP CoASHG° 40 kJ/mol

Glucose metabolized via glycolysis produces two molecules of pyruvate and thus two molecules of acetyl-CoA, which can enter the TCA cycle. Combining glycolysis and the TCA cycle gives the net reaction shown:

Glucose 2 H2O 10 NAD 2 [FAD] 4 ADP 4 Pi 34

6 CO2 10 NADH 10 H 2 [FADH2] 4 ATP

All six carbons of glucose are liberated as CO2, and a total of four molecules of ATP are formed thus far in substrate-level phosphorylations. The 12 reduced coenzymes produced up to this point can eventually produce a maximum of 34 molecules of ATP in the electron transport and oxidative phosphorylation pathways. A stoichiometric relationship for these subsequent processes is

NADH H 12 O2 3 ADP 3 Pi 34 NAD 3 ATP 4 H2O [FADH2] 12 O2 2 ADP 2 Pi 34 [FAD] 2 ATP 3 H2O

Thus, a total of 3 ATP per NADH and 2 ATP per FADH2 may be produced through the processes of electron transport and oxidative phosphorylation.

The Fate of the Carbon Atoms of Acetyl-CoA in the TCA Cycle

It is instructive to consider how the carbon atoms of a given acetate group are routed through several turns of the TCA cycle. As shown in Figure 20.21, neither of the carbon atoms of a labeled acetate unit is lost as CO2 in the first turn of the cycle. The CO2 evolved in any turn of the cycle derives from the carboxyl groups of the oxaloacetate acceptor (from the previous turn), not from incoming acetyl-CoA. On the other hand, succinate labeled on one end from the original labeled acetate forms two different labeled oxaloacetates. The carbonyl carbon of acetyl-CoA is evenly distributed between the two carboxyl carbons of oxaloacetate, and the labeled methyl carbon of incoming acetyl-CoA ends up evenly distributed between the methylene and carbonyl carbons of oxaloacetate.

When these labeled oxaloacetates enter a second turn of the cycle, both of the carboxyl carbons are lost as CO2, but the methylene and carbonyl carbons survive through the second turn. Thus, the methyl carbon of a labeled acetyl-CoA survives two full turns of the cycle. In the third turn of the cycle, one-half of the carbon from the original methyl group of acetyl-CoA has become one of the carboxyl carbons of oxaloacetate and is thus lost as CO2. In the fourth turn of the cycle, further “scrambling” results in loss of half of the remaining labeled carbon (one-fourth of the original methyl carbon label of acetyl-CoA), and so on.

It can be seen that the carbonyl and methyl carbons of labeled acetyl-CoA have very different fates in the TCA cycle. The carbonyl carbon survives the first turn intact but is completely lost in the second turn. The methyl carbon

660 Chapter 20 ● The Tricarboxylic Acid Cycle

(a) Fate of the carboxyl carbon of acetate unit

O

S CoA

O

HO

Oxaloacetate

Citrate

HO

Malate

1st turn

HO

Isocitrate

Fumarate

(CO2)

α -Ketoglutarate

Succinate

Succinyl–CoA

(CO2)

(b) Fate of methyl carbon of acetate unit

O

S CoA

O

HO

Oxaloacetate

Citrate

HO

Malate

1st turn

HO

Isocitrate

Fumarate

(CO2)

α -Ketoglutarate

Succinate

Succinyl–CoA

(CO2)

	O
	S	CoA
O
	HO
Oxaloacetate		Citrate
HO
Malate	2nd turn		HO
			Isocitrate
Fumarate			1
			/2
			(CO2)
			α -Ketoglutarate
Succinate
	Succinyl–CoA		1
			/2
			(CO2)
			All labelled carboxyl
			carbon removed by
	O		these two steps
	S	CoA

O

HO

Oxaloacetate

Citrate

HO

Malate

2nd turn

HO

Isocitrate

Fumarate

(CO2)

α -Ketoglutarate

Succinate

Succinyl–CoA

(CO2)