Biochemistry, 4th Edition P61 docx

© Richard Cummins/CORBISUnder aerobic conditions, pyruvate from glycolysis is converted to acetyl-coenzyme A acetyl-CoA and oxidized to CO2 in the tricarboxylic acid TCA cycle also calle

© Richard Cummins/CORBIS

Under aerobic conditions, pyruvate from glycolysis is converted to acetyl-coenzyme

A (acetyl-CoA) and oxidized to CO2 in the tricarboxylic acid (TCA) cycle (also

called the citric acid cycle) The electrons liberated by this oxidative process are

passed via NADH and FADH2through an elaborate, membrane-associated

electron-transport pathwayto O2, the final electron acceptor Electron transfer is coupled to

creation of a proton gradient across the membrane Such a gradient represents an

energized state, and the energy stored in this gradient is used to drive the synthesis

of many equivalents of ATP

ATP synthesis as a consequence of electron transport is termed oxidative

phosphorylation; the complete process is diagrammed in Figure 19.1 Aerobic

pathways permit the production of 30 to 38 molecules of ATP per glucose oxidized

Although two molecules of ATP come from glycolysis and two more directly out of

the TCA cycle, most of the ATP arises from oxidative phosphorylation Specifically,

reducing equivalents released in the oxidative reactions of glycolysis, pyruvate

de-carboxylation, and the TCA cycle are captured in the form of NADH and

enzyme-bound FADH2, and these reduced coenzymes fuel the electron-transport pathway

and oxidative phosphorylation

Complete oxidation of glucose to CO2involves the removal of 24 electrons—that

is, it is a 24-electron oxidation In glycolysis, 4 electrons are removed as NADH, and

4 more exit as two more NADH in the decarboxylation of two molecules of pyruvate

to two acetyl-CoA (Figure 19.1) For each acetyl-CoA oxidized in the TCA cycle,

8 more electrons are removed (as three NADH and one FADH2):

H3CCOO 2H2O H⎯⎯→ 2CO2 8H

In the electron-transport pathway these 8 electrons combine with oxygen to form

water:

8H 2O2⎯⎯→ 4H2O

So, the net reaction for the TCA cycle and electron transport pathway is

H3CCOO 2O2 H⎯⎯→ 2CO2 2H2O

As German biochemist Hans Krebs showed in the 1930s, the eight-electron

oxi-dation of acetate by the TCA cycle is accomplished with the help of oxaloacetate

(In his honor, the TCA cycle is often referred to as the Krebs cycle.) Beginning with

acetate, a series of five reactions produces two molecules of CO2, with four electrons

extracted in the form of NADH and four electrons passed to oxaloacetate to

pro-duce a molecule of succinate The pathway becomes a cycle by three additional

re-actions that accomplish a four-electron oxidation of succinate back to oxaloacetate

This special trio of reactions is used repeatedly in metabolism: first, oxidation of a single bond

to a double bond, then addition of the elements of water across the double bond, and finally

A time-lapse photograph of a ferris wheel at night Aerobic cells use a metabolic wheel—the tricarboxylic acid cycle—to generate energy by acetyl-CoA oxidation.

Thus times do shift, each thing his turn does hold;

New things succeed, as former things grow old.

Robert Herrick

Hesperides (1648), “Ceremonies for Christmas Eve”

KEY QUESTIONS

19.1 What Is the Chemical Logic of the TCA Cycle?

19.2 How Is Pyruvate Oxidatively Decarboxylated

to Acetyl-CoA?

19.3 How Are Two CO 2 Molecules Produced from Acetyl-CoA?

19.4 How Is Oxaloacetate Regenerated to Complete the TCA Cycle?

19.5 What Are the Energetic Consequences

of the TCA Cycle?

19.6 Can the TCA Cycle Provide Intermediates for Biosynthesis?

19.7 What Are the Anaplerotic, or “Filling Up,” Reactions?

19.8 How Is the TCA Cycle Regulated?

19.9 Can Any Organisms Use Acetate as Their Sole Carbon Source?

ESSENTIAL QUESTION

The glycolytic pathway converts glucose to pyruvate and produces two molecules

of ATP per glucose—only a small fraction of the potential energy available from

glucose Under anaerobic conditions, pyruvate is reduced to lactate in animals and

to ethanol in yeast, and much of the potential energy of the glucose molecule

remains untapped In the presence of oxygen, however, a much more interesting

and thermodynamically complete story unfolds

How is pyruvate oxidized under aerobic conditions, and what is the chemical

logic that dictates how this process occurs?

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564 Chapter 19 The Tricarboxylic Acid Cycle

oxidation of the resulting alcohol to a carbonyl We will see it again in fatty acid oxidation

(see Chapter 23), in reverse in fatty acid synthesis (see Chapter 24), and in amino acid synthesis and breakdown (see Chapter 25)

19.1 What Is the Chemical Logic of the TCA Cycle?

The entry of new carbon units into the cycle is through acetyl-CoA This entry metabolite can be formed either from pyruvate (from glycolysis) or from oxidation

of fatty acids (discussed in Chapter 23) Transfer of the two-carbon acetyl group from acetyl-CoA to the four-carbon oxaloacetate to yield six-carbon citrate is cat-alyzed by citrate synthase A dehydration–rehydration rearrangement of citrate yields isocitrate Two successive decarboxylations produce -ketoglutarate and then

succinyl-CoA, a CoA conjugate of a four-carbon unit Several steps later, oxaloac-etate is regenerated and can combine with another two-carbon unit of acetyl-CoA Thus, carbon enters the cycle as acetyl-CoA and exits as CO2 In the process, meta-bolic energy is captured in the form of ATP, NADH, and enzyme-bound FADH2 (symbolized as [FADH2])

The TCA Cycle Provides a Chemically Feasible Way of Cleaving

a Two-Carbon Compound

The cycle shown in Figure 19.2 at first appears to be a complicated way to oxidize acetate units to CO2, but there is a chemical basis for the apparent complexity Oxidation of an acetyl group to a pair of CO2molecules requires COC cleavage:

CHCOO⎯⎯→ CO  CO

O2

H2O

Acetyl-CoA

Citric acid cycle -Ketoglutarate

Isocitrate Citrate

Succinyl-CoA

Fumarate

Oxaloacetate

Malate

Succinate

[FADH2]

FADH

2

+

Electron transport

Oxidative phosphorylation

Proton gradient

Mitochondrial matrix

Intermembrane space

+

H +

H + H +

H +

H +

H +

H + H +

e –

e –

e –

GTP GDP Pi

Pi

NADH

NADH

NADH

NADH

e –

Pyruvate

Glucose

Glycolysis

NAD+

NADH

NAD+

NADH

(a)

(b)

(c)

ATP

ADP+Pi

tricar-boxylic acid (TCA) cycle (c) Electrons liberated in this oxidation flow through the

electron-transport chain and drive the synthesis of ATP in oxidative phosphory-lation In eukaryotic cells, this overall process occurs in mitochondria.

Pyruvate dehydrogenase

Citrate synthase

2 Aconitase

3 Isocitrate dehydrogenase

-Ketoglutarate

dehydrogenase

4 5

Nucleoside diphosphate kinase

Succinyl-CoA synthetase

Succinate

dehydrogenase

Fumarase

6

7

8

Malate dehydrogenase

Isocitrate

H2C COO–

HC OH COO–

C O

Acetyl-CoA

O

Pyruvate

C

H3C

H3C

H2C

C O

O–

O From glycolysis

From-oxidation of fatty acids

Oxaloacetate

COO–

COO–

H2C COO–

2 C COO–

Citrate

-Ketoglutarate

C

C

SCoA

Succinyl-CoA

O

O

Succinate

FADH2 FAD

C

C

Fumarate

H

H

HO

H2C COO–

H2C

H2C COO–

H2C

H2C COO–

COO–

H2C COO–

Malate

TRICARBOXYLIC ACID

CYCLE (citric acid cycle, Krebs cycle, TCA cycle)

H2O

H2O

COO–

–OOC

ATP

Pi

ADP

CoASH

NADH+

NAD +

NAD + NAD+

NAD+

CoASH

CoASH

CO2

CoA

NADH+

NADH+ NADH+ H +

CO2

CO2

H +

H +

H+

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566 Chapter 19 The Tricarboxylic Acid Cycle

In many instances, COC cleavage reactions in biological systems occur between car-bon atoms  and  to a carbonyl group:

A good example of such a cleavage is the fructose bisphosphate aldolase reaction (see Figure 18.12)

Another common type of COC cleavage is -cleavage of an -hydroxyketone:

(We see this type of cleavage in the transketolase reaction described in Chapter 22.) Neither of these cleavage strategies is suitable for acetate It has no -carbon, and

the second method would require hydroxylation—not a favorable reaction for ac-etate Instead, living things have evolved the clever chemistry of condensing acetate with oxaloacetate and then carrying out a -cleavage The TCA cycle combines this

-cleavage reaction with oxidation to form CO2, regenerate oxaloacetate, and cap-ture the liberated metabolic energy in NADH and ATP

19.2 How Is Pyruvate Oxidatively Decarboxylated

to Acetyl-CoA?

Pyruvate produced by glycolysis is a significant source of acetyl-CoA for the TCA cy-cle Because, in eukaryotic cells, glycolysis occurs in the cytoplasm, whereas the TCA cycle reactions and all subsequent steps of aerobic metabolism take place in the mi-tochondria, pyruvate must first enter the mitochondria to enter the TCA cycle The oxidative decarboxylation of pyruvate to acetyl-CoA

Pyruvate CoA  NAD⎯⎯→ acetyl-CoA  CO2 NADH

is the connecting link between glycolysis and the TCA cycle The reaction is cat-alyzed by pyruvate dehydrogenase, a multienzyme complex

The pyruvate dehydrogenase complex (PDC) is formed from multiple copies

of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) All are involved in the conversion of

pyru-vate to acetyl-CoA The active sites of all three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme, and so on, without diffusion of substrates and products through the solution

Eukaryotic PDC, one of the largest-known multienzyme complexes (with a di-ameter of approximately 500 Å) is a 9.5-megadalton assembly organized around an icosahedral 60-mer of E2 subunits, with 30 E1 heterotetramers and 12 homodimers

of E3 (Figure 19.3) Eukaryotic PDC also contains an E3-binding protein (E3BP)

that is required to bind E3 to the PDC Trimeric units of E2 form the 20 vertices of the icosahedron, with E3BP bound in each of the 12 faces The E2 subunits each carry a lipoic acid moiety covalently linked to a lysine residue Flexible linker seg-ments in E2 and E3BP impart the flexibility that allows the lipoic acid groups to visit all three active sites during catalysis

The pyruvate dehydrogenase reaction (Figure 19.4) is a tour de force of mecha-nistic chemistry, involving as it does a total of three enzymes and five different coen-zymes The first step of this reaction, decarboxylation of pyruvate and transfer of the

C

O OH

C

Cleavage

C

O

C C

Cleavage

acetyl group to lipoic acid, depends on accumulation of negative charge on the

transferred two-carbon fragment, as facilitated by the quaternary nitrogen on the

thiazolium group of thiamine pyrophosphate (TPP) As shown in Figure 19.5, this

cationic imine nitrogen plays two distinct roles in TPP-catalyzed reactions:

1 It provides electrostatic stabilization of the thiazole carbanion formed upon

re-moval of the C-2 proton (The sp2hybridization and the availability of vacant

d orbitals on the adjacent sulfur probably also facilitate proton removal at C-2.)

2 TPP attack on pyruvate leads to decarboxylation The TPP cationic imine

nitro-gen can act as an effective electron sink to stabilize the negative charge that must

develop on the carbon that has been attacked This stabilization takes place by

resonance interaction through the double bond to the nitrogen atom

This resonance-stabilized intermediate can be protonated to give

hydroxyethyl-TPP.The reaction of hydroxyethyl-TPP with the oxidized form of lipoic acid yields

the energy-rich acetyl-thiol ester of reduced lipoic acid through oxidation of the

hydroxyl-carbon of the two-carbon substrate unit Nucleophilic attack by CoA on

the carbonyl carbon (a characteristic feature of CoA chemistry) results in transfer of

the acetyl group from lipoic acid to CoA The subsequent oxidation of lipoic acid is

catalyzed by the FAD-dependent dihydrolipoyl dehydrogenase, and NADis reduced

(E3 not shown) E1 subunits (yellow) are joined to the E2 core (green) by linkers (blue).(Adapted from Zhou, Z H., McCarthy, D B., O’Connor, C M., Reed, L J., and Stoops, J K., 2001 The remarkable structural and functional organization of the

eukaryotic pyruvate dehydrogenase complexes Proc Natl Acad

Sci U S A 98:14802–14807 Figure courtesy of Z Hong Zhou.)

CH3 C O

CH3 C COO–

O

CH3

TPP

CH3C O

[FAD]

SCoA

Protein

S S

CoASH

NAD +

NADH

Pyruvate

Thiamine pyrophosphate

Hydroxyethyl TPP (HETPP)

Pyruvate loses CO2 and

HETPP is formed

Hydroxyethyl group is transferred to lipoic acid and oxidized to form acetyl dihydrolipoate

Acetyl group is transferred

to CoA

Lipoic acid

is reoxidized

Pyruvate dehydrogenase

Dihydrolipoyl transacetylase (dihydrolipoamide acetyltransferase)

Dihydrolipoyl dehydrogenase (dihydrolipoamide dehydrogenase)

Lipoic acid

CO2

3

3

4

occurs with formation of hydroxyethyl-TPP (step 1) Transfer of the two-carbon unit to lipoic acid in step 2 is

fol-lowed by formation of acetyl-CoA in step 3 Lipoic acid is reoxidized in step 4 of the reaction.

568 Chapter 19 The Tricarboxylic Acid Cycle

A DEEPER LOOK

The Coenzymes of the Pyruvate Dehydrogenase Complex

Coenzymes are small molecules that bring unique chemistry to

en-zyme reactions Five coenen-zymes are used in the pyruvate

dehydro-genase reaction

Thiamine Pyrophosphate

trans-ketolase reaction, see Chapter 22)

NH2

N

N

H3C

H C

H N+

H3C HC

H

H C

+

NH2 N N

H3C

H C H

H C H

H C

O

O–

P O O–

H3C

ATP

Thiamine (vitamin B 1 )

TPP Synthetase

Thiamine pyrophosphate (TPP)

Acidic proton AMP

O

C O

N

NH2

+

H

O H H OH OH H

CH2 P

–O

P O

H

O H H OH OH H

N N

NH2 O

C O

N

NH2 H

H

H

1 2 3 4

6 5

O

Nicotinamide

(oxidized form)

Nicotinamide

(reduced form)

Hydride ion,

H –

pro-R pro-S

AMP

NADP+ contains a Pi

on this 2 -hydroxyl

The Nicotinamide Coenzymes

involving these coenzymes are two-electron

transfers

H3C N

H

N

N O

CH2

HCOH

CH2 O P

O P

OH OH

H H

N

N

NH2

D -Ribitol

O

HCOH HCOH

N NH O R

8 7 6

9 9a

5a 10

4a

2

1 10a

H+, e– H+, e–

N NH O R

N N O R

O H

pKa≅ 8.4

N N O R

H

H O

H+, e–

H+, e–

–

H–+H+

H–+H+

Isoalloxazine

AMP

FAD or FMN

FADH or FMNH

Oxidized form

max = 450 nm

(yellow)

Semiquinone form

max = 570 nm

(blue)

Semiquinone anion

max = 490 nm (red)

FADH 2 or FMNH 2

Reduced form (colorless)

(a)

(b)

Flavin coenzymes can exist in any of three redox states, and this allows flavin coenzymes to participate in one-electron transfer and two-electron transfer reactions Partly because

of this, flavoproteins catalyze many reactions in biological systems and work with many electron acceptors and donors Because the ribityl group is not a true pentose sugar (it is

a sugar alcohol) and is not joined to riboflavin in a glyco-sidic bond, the molecule is not truly a “nucleotide” and the

terms flavin mononucleotide and dinucleotide are incorrect.

Nonetheless, these designations are so deeply ingrained in common biochemical usage that the erroneous nomencla-ture persists

Continued

570 Chapter 19 The Tricarboxylic Acid Cycle

A DEEPER LOOK

The Coenzymes of the Pyruvate Dehydrogenase Complex (cont’d)

Coenzyme A

The two main functions of CoA are:

1 Activation of acyl groups for transfer by nucleophilic attack

proton

The reactive sulfhydryl group on CoA mediates both of these

func-tions The sulfhydryl group forms thioester linkages with acyl groups.

The two main functions of CoA are illustrated in the citrate synthase

reaction (see Figure 19.6)

β

O C

CH2 SH

CH2 NH

O C

CH2

CH2 NH

HCOH

H3C C CH3

CH2 O

–O O

–O O

CH2 O

H H

PO3–

N

N N

N

NH2

3'

-Mercaptoethylamine

Pantothenic acid

3 ⴕ,5ⴕ–ADP

CHCH2CH2CH2CH2C

H2C

O

N

CH HN

O H

C

H2C

CH2

CHCH2CH2CH2CH2C

O–

O

Lipoic acid, oxidized form Reduced form

Lipoyllysine (lipoamide)

Lipoic Acid

Lipoic acid functions to couple acyl-group transfer and electron

acid is covalently bound to relevant enzymes through amide bond

19.3 How Are Two CO2Molecules Produced from Acetyl-CoA?

The Citrate Synthase Reaction Initiates the TCA Cycle

The first reaction within the TCA cycle, the one by which carbon atoms are

intro-duced, is the citrate synthase reaction (Figure 19.6) Here acetyl-CoA reacts with

ox-aloacetate in a Perkin condensation (a carbon–carbon condensation between a ketone

or aldehyde and an ester) The acyl group is activated in two ways in an acyl-CoA

mol-ecule: The carbonyl carbon is activated for attack by nucleophiles, and the Ccarbon

is more acidic and can be deprotonated to form a carbanion The citrate synthase

re-action depends upon the latter mode of activation As shown in Figure 19.6, a general

base on the enzyme accepts a proton from the methyl group of acetyl-CoA, producing

a stabilized -carbanion of acetyl-CoA This strong nucleophile attacks the -carbonyl

of oxaloacetate, yielding citryl-CoA This part of the reaction has an equilibrium

constant near 1, but the overall reaction is driven to completion by the subsequent

Lipoamide

C

CH3

COO–

O S

N R"

R

R'

B +

S N R"

R

R'

S N R"

R

R' + C

CH3

C OH

S N R"

R

R' + C

CH3 OH

S N R"

R

R'

C

CH3

OH H+

S N R"

R

R'

C

CH3 OH H

E

H

– –

–

:B C

CH3

S S

Lipoamide

H:B

C

CH3

CH3 O

O H

S SH

_

CH3 O

CoA S C CoA S H:

+

CO2

–

–

:

N

N

N

Lipoamide

S SH

Lipoamide

S SH

Lipoamide

SH SH

Resonance-stabilized carbanion on substrate

Hydroxyethyl-TPP

Pyruvate

steps of the pyruvate dehydrogenase complex reaction.

O

H

H

O

B

B

H

H

C COO–

COO–

Oxaloacetate

H2C

C SCoA O

H2C COO–

Citryl-CoA

H2C

H2C COO–

COO–

Citrate

pro-S arm

pro-R arm

+

E

CoA

E

acetyl-CoA The mechanism involves nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis.

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572 Chapter 19 The Tricarboxylic Acid Cycle

hydrolysis of the high-energy thioester to citrate and free CoA The overall G° is

31.4 kJ/mol, and under standard conditions the reaction is essentially irreversible Although the mitochondrial concentration of oxaloacetate is very low (much less than 1

forward

Citrate Synthase Is a Dimer Citrate synthase in mammals is a dimer of 49-kD sub-units (Table 19.1) On each subunit, oxaloacetate and acetyl-CoA bind to the active site, which lies in a cleft between two domains and is surrounded mainly by -helical

segments (Figure 19.7) Binding of oxaloacetate induces a conformational change that facilitates the binding of acetyl-CoA and closes the active site so that the reactive carbanion of acetyl-CoA is protected from protonation by water

NADH Is an Allosteric Inhibitor of Citrate Synthase Citrate synthase is the first step in this metabolic pathway, and as stated the reaction has a large negative G°.

As might be expected, it is a highly regulated enzyme NADH, a product of the TCA cycle, is an allosteric inhibitor of citrate synthase, as is succinyl-CoA, the product of the fifth step in the cycle (and an acetyl-CoA analog)

Citrate Is Isomerized by Aconitase to Form Isocitrate

Citrate itself poses a problem: It is a poor candidate for further oxidation because

it contains a tertiary alcohol, which could be oxidized only by breaking a carbon–carbon bond An obvious solution to this problem is to isomerize the ter-tiary alcohol to a secondary alcohol, which the cycle proceeds to do in the next step

Citrate is isomerized to isocitrate by aconitase in a two-step process involving

aconitate as an intermediate (Figure 19.8) In this reaction, the elements of water are first abstracted from citrate to yield aconitate, which is then rehydrated with

HO and HOO adding back in opposite positions to produce isocitrate The net ef-fect is the conversion of a tertiary alcohol (citrate) to a secondary alcohol (iso-citrate) Oxidation of the secondary alcohol of isocitrate involves breakage of a COH bond, a simpler matter than the COC cleavage required for the direct oxi-dation of citrate

Inspection of the citrate structure shows a total of four chemically equivalent

hy-drogens, but only one of these—the pro-R H atom of the pro-R arm of citrate—is

ab-stracted by aconitase, which is quite stereospecific Formation of the double bond of aconitate following proton abstraction requires departure of hydroxide ion from the C-3 position Hydroxide is a relatively poor leaving group, and its departure is facili-tated in the aconitase reaction by coordination with an iron atom in an iron–sulfur cluster

dehydrogenase complex

G values from Newsholme, E A., and Leech, A R., 1983 Biochemistry for the Medical Sciences New York:Wiley.

TABLE 19.1 The Enzymes and Reactions of the TCA Cycle

CoASH

here, citrate is shown in blue, and CoA is red (Top: pdb id

 1CTS; bottom:pdb id  2CTS.)