Voet D., Voet Ju.G. Biochemistry

Подождите немного. Документ загружается.

Occasionally, 2,3-BPG dissociates from the enzyme (Fig.

17-18; Step 5), leaving it in an inactive form. Trace amounts

of 2,3-BPG must therefore always be available to regener-

ate the active phosphoenzyme by the reverse reaction.

b. Glycolysis Influences Oxygen Transport

2,3-BPG specifically binds to deoxyhemoglobin and

thereby alters the oxygen affinity of hemoglobin (Section

10-1D). The concentration of 2,3-BPG in erythrocytes is

much higher (⬃5 mM) than the trace amounts required for

its use as a primer of PGM. Erythrocytes synthesize and

degrade 2,3-BPG by a detour from the glycolytic pathway,

diagrammed in Fig. 17-19. Bisphosphoglycerate mutase

catalyzes the transfer of a phosphoryl group from C1 to C2

of 1,3-BPG.The resulting 2,3-BPG is hydrolyzed to 3PG by

2,3-bisphosphoglycerate phosphatase. The rate of glycoly-

sis affects the oxygen affinity of hemoglobin through the

mediation of 2,3-BPG. Consequently, inherited defects of

Section 17-2. The Reactions of Glycolysis 611

Figure 17-18 Proposed reaction mechanism for

phosphoglycerate mutase. The active form of the enzyme

contains a phospho-His residue at the active site. (1) Formation

of an enzyme–substrate complex; (2) transfer of the enzyme-

bound phosphoryl group to the substrate; (3) rephosphorylation

Phosphoenzyme

–

OPO

2–

–

His

–

OPO

2–

His

OPO

2–

2,3-BPG•Enzyme

complex

–

OPO

2–

–

His

–

His

3PG

3PG•Phosphoenzyme

complex

2PG•Phosphoenzyme

complex

2PG

Dephosphoenzyme

(inactive)

2,3-BPG

of the enzyme by the other phosphoryl group of the substrate;

and (4) release of product regenerating the active

phosphoenzyme. (5) Occasionally, 2,3-BPG dissociates from the

enzyme, leaving it in an inactive, dephospho form that must be

rephosphorylated by the reverse reaction.

JWCL281_c17_593-637.qxd 2/26/10 1:38 PM Page 611

glycolysis in erythrocytes alter the capacity of the blood to

transport oxygen (Fig. 17-20). For example, the concentra-

tion of glycolytic intermediates in hexokinase-deficient

erythrocytes is less than normal because hexokinase cat-

alyzes the first reaction of glycolysis. This results in a di-

minished 2,3-BPG concentration and therefore in increased

hemoglobin oxygen affinity. Conversely, pyruvate kinase

deficiency decreases hemoglobin oxygen affinity through

the increase of 2,3-BPG resulting from the blockade of the

last reaction in glycolysis. Thus, although erythrocytes,

which lack nuclei and other organelles, have but a minimal

metabolism, this metabolism is physiologically significant.

I. Enolase: Second “High-Energy”

Intermediate Formation

In Reaction 9 of glycolysis, 2PG is dehydrated to phospho-

enolpyruvate (PEP) in a reaction catalyzed by enolase:

The enzyme forms a complex with a divalent cation such as

2

before the substrate is bound. A second divalent

metal ion then binds to the enzyme.As is mentioned in Sec-

tion 17-1A, fluoride ion inhibits glycolysis, resulting in the

accumulation of 2PG and 3PG. It does so by strongly in-

hibiting enolase in the presence of P



and P

form a

tightly bound complex with the Mg

2

at the enzyme’s ac-

tive site, blocking substrate binding and thereby inactivat-

ing the enzyme. Enolase’s substrate, 2PG, therefore builds

up and, as it does so, is equilibrated with 3PG by PGM.

a. Catalytic Mechanism of Enolase

The dehydration (elimination of H

O) catalyzed by eno-

lase might occur in one of three ways (Fig. 16-9a): (1) The

group at C3 can leave first, generating a carbocation¬OH

2-Phosphoglycerate

(2PG)

–

OPO

–

Phosphoenolpyruvate

(PEP)

–

C + H

OPO

–

enolase

at C3; (2) the C2 proton can leave first, generating a carban-

ion at C2; or (3) the reaction can be concerted. Isotope

exchange studies by Paul Boyer demonstrated that the C2

proton of 2PG exchanges with solvent 12 times faster than

the rate of PEP formation. However, the C3 oxygen ex-

changes with solvent at a rate roughly equivalent with the

overall reaction rate.This suggests the following mechanism

(Fig. 17-21):

Step 1 Rapid carbanion formation at C2 facilitated by a

general base on the enzyme. The abstracted proton can

readily exchange with the solvent, accounting for its ob-

served rapid exchange rate.

Step 2 Rate-limiting elimination of the group at

C3.This is consistent with the slow rate of exchange of this

hydroxyl group with solvent.

¬OH

612 Chapter 17. Glycolysis

Figure 17-19 The pathway for the synthesis

and degradation of 2,3-BPG in erythrocytes is a

detour from the glycolytic pathway.

Figure 17-20 The oxygen-saturation curves of hemoglobin in

normal erythrocytes (red curve) and in those from patients with

hexokinase deficiency (green) and with pyruvate kinase

deficiency (purple). [After Delivoria-Papadopoulos, M., Oski,

F.A., and Gottlieb,A.J., Science 165, 601 (1969).]

3-Phosphoglycerate

2-Phosphoglycerate

1,3-Bisphosphoglycerate

2,3-Bisphospho-

glycerate

(2,3-BPG)

Glyceraldehyde 3-phosphate

GAPDH

PGK

PGM

bisphosphoglycerate

mutase

2,3-bisphosphoglycerate

phosphatase



CHOPO

2

OPO

2

0 102030405060

(torr)

Hexokinase

deficient

Pyruvate kinase

deficient

Normal

erythrocytes

100

Oxygen saturation (%)

JWCL281_c17_593-637.qxd 2/26/10 1:38 PM Page 612

⫺

⫹

2⫹

⫺

2⫹

Lys 345

Lys 396

⫺

⫹

1 fast

2-Phosphoglycerate (2 PG)

⫺

⫹

⫺

H H

⫹

2 slow

Delocalized carbanion intermediate

⫹

rapid

exchange

O HOH

Glu 211

Phosphoenolpyruvate (PEP)

⫺

The enolase reaction (Fig. 17-21) is of mechanistic inter-

est because it involves the abstraction of the decidedly

nonacidic proton at C2 (pK ⬎ 30), followed by the elimina-

tion of an OH

⫺

ion, which is a poor leaving group. The X-

ray structure of yeast enolase in complex with two Mg

2⫹

ions and an equilibrium mixture of 2PG and PEP (eno-

lase’s substrate and product), determined by George Reed

and Ivan Rayment, reveals that enolase binds 2PG in an in-

tricate complex that involves both Mg

2⫹

ions. Mutagenic

and enzymological studies indicate that the reaction in-

volves the Lys 345 side chain functioning as a general base

and the Glu 211 side chain functioning as a general acid.

Lys 396 and the two Mg

2⫹

ions are thought to stabilize the

increased negative charge that develops on the carboxylate

ion in the delocalized carbanion intermediate.

J. Pyruvate Kinase: Second ATP Generation

In Reaction 10 of glycolysis, its final reaction, pyruvate ki-

nase (PK) couples the free energy of PEP hydrolysis to the

synthesis of ATP to form pyruvate:

Phosphoenolpyruvate

(PEP)

–

C + ADP

OPO

–

Pyruvate

–

C + ATP

pyruvate

kinase (PK)

Section 17-2. The Reactions of Glycolysis 613

Figure 17-21 Proposed reaction mechanism of enolase. (1)

Rapid formation of a carbanion by removal of a proton at C2 by

Lys 345 acting as a general base; this proton can rapidly exchange

with the solvent. (2) Slow elimination of H

O to form phospho-

enolpyruvate with general acid catalysis by Glu 211; the C3 oxy-

gen of the substrate can exchange with solvent only as rapidly as

this step occurs.

JWCL281_c17_593-637.qxd 6/30/10 10:54 AM Page 613

a. Catalytic Mechanism of PK

The PK reaction, which requires the participation of

both monovalent (K

⫹

) and divalent (Mg

2⫹

) cations, occurs

as follows (Fig. 17-22):

Step 1 A ␤-phosphoryl oxygen of ADP nucleophilically

attacks the PEP phosphorus atom, thereby displacing

enolpyruvate and forming ATP. This reaction conserves

the free energy of PEP hydrolysis.

Step 2 Enolpyruvate converts to pyruvate.This enol–keto

tautomerization is sufficiently exergonic to drive the coupled

endergonic synthesis of ATP (Section 16-4Ba).

We can now see the “logic” of the enolase reaction. The

standard free energy of hydrolysis of 2PG (⌬G°¿) is only

⫺17.6 kJ ⴢ mol

⫺1

, which is insufficient to drive ATP synthe-

sis (⌬G°¿ ⫽ 30.5 kJ ⴢ mol

⫺1

for ATP synthesis from ADP

and P

).The dehydration of 2PG results in the formation of

a “high-energy” compound capable of such synthesis [the

standard free energy of hydrolysis of PEP is ⫺61.9

kJ ⴢ mol

⫺1

(Fig. 16-25)]. In other words, PEP is a “high-

energy” compound, 2PG is not.

3 FERMENTATION: THE ANAEROBIC

FATE OF PYRUVATE

For glycolysis to continue, NAD

⫹

, which cells have in lim-

ited quantities, must be recycled after its reduction to

NADH by GAPDH (Fig. 17-3; Reaction 6). In the presence

of oxygen, the reducing equivalents of NADH are passed

into the mitochondria for reoxidation (Chapter 22). Under

anaerobic conditions, on the other hand, the NAD

⫹

is re-

plenished by the reduction of pyruvate in an extension of

the glycolytic pathway.Two processes for the anaerobic re-

plenishment of NAD

⫹

are homolactic and alcoholic fer-

mentation, which occur in muscle and yeast, respectively.

A. Homolactic Fermentation

In muscle, particularly during vigorous activity when the

demand for ATP is high and oxygen has been depleted, lac-

tate dehydrogenase (LDH) catalyzes the oxidation of

NADH by pyruvate to yield NAD

⫹

and lactate. This reac-

tion is often classified as Reaction 11 of glycolysis:

LDH, as do other NAD

⫹

-requiring enzymes, catalyzes its

reaction with absolute stereospecificity: The pro-R (A-side)

hydrogen at C4 of NADH is stereospecifically transferred

to the re face of pyruvate at C2 to form

L- (or S-) lactate.

This regenerates NAD

⫹

for participation in the GAPDH

reaction.The hydride transfer to pyruvate is from the same

face of the nicotinamide ring as that to acetaldehyde in the

alcohol dehydrogenase reaction (Section 13-2A) but from

the opposite (si) face of the nicotinamide ring as that to

GAP in the GAPDH reaction (Section 17-2F).

Mammals have two different types of LDH subunits, the

M type and the H type, which together form five tetrameric

isozymes: M

H, M

,MH

, and H

. Although these

hybrid forms occur in most tissues, the H-type subunit pre-

dominates in aerobic tissues such as heart muscle, while the

M-type subunit predominates in tissues that are subject to

anaerobic conditions such as skeletal muscle and liver. H

–

NADH

NAD

Pyruvate

L-Lactate

–

HHO

lactate

dehydrogenase (LDH)

614 Chapter 17. Glycolysis

Figure 17-22 Mechanism of the reaction catalyzed by pyruvate

kinase. (1) Nucleophilic attack of an ADP ␤-phosphoryl oxygen

atom on the phosphorus atom of PEP to form ATP and

–

ATP

Phosphoenol-

pyruvate (PEP)

ADP

–

O P

O Adenosine

–

Enolpyruvate

––

C C

Pyruvate

–

ΔG°´

⫽ ⫹14.6 kJ

•

mol

⫺1

ΔG°´

⫽ ⫺46 kJ

•

mol

⫺1

Overall ΔG°´

⫽ ⫺31.4 kJ

•

mol

⫺1

enolpyruvate; and (2) tautomerization of enolpyruvate to

pyruvate.

JWCL281_c17_593-637.qxd 6/3/10 8:37 AM Page 614

LDH has a low K

for pyruvate and is allosterically inhib-

ited by high levels of this metabolite, whereas the M

isozyme has a higher K

for pyruvate and is not inhibited

by it.The other isozymes have intermediate properties that

vary with the ratio of their two types of subunits. It has

therefore been proposed, although not without disagree-

ment, that H-type LDH is better adapted to function in the

oxidation of lactate to pyruvate, whereas M-type LDH is

more suited to catalyze the reverse reaction.

The X-ray structure of porcine H

LDH in complex with

S-lac-NAD

⫹

(a bisubstrate analog in which atom C3 of lac-

tate is covalently linked to nicotinamide atom C5 of NAD

⫹

via a CH

group) was determined by Michael Rossmann

(Fig. 17-23; he also determined the X-ray structure of dog-

fish M

LDH shown in Fig. 8-54a). Lactate atom O2, its hy-

droxyl oxygen, is hydrogen bonded to the side chains of

both Arg 109 and His 195, whereas the lactate carboxyl

group at C1 is doubly hydrogen bonded to the side chain of

Arg 171. On the basis of this structure and extensive enzy-

mological evidence, Rossmann proposed the following

mechanism for pyruvate reduction by LDH (Fig. 17-24):

The pro-R hydride is transferred from C4 of NADH’s

nicotinamide ring to C2 of pyruvate with the concomitant

transfer of a proton from the imidazolium moiety of His 195

to pyruvate O2, thereby yielding NAD

⫹

and lactate. The

proton transfer is facilitated by repulsive interactions with

the closely associated positively charged side chain of Arg

109. These interactions also serve to properly orient the

pyruvate, as does the salt bridge that the pyruvate carboxyl

group forms with the side chain of Arg 171.

The overall process of anaerobic glycolysis in muscle

can be represented:

Much of the lactate, the end product of anaerobic glycolysis,

is exported from the muscle cell via the blood to the liver,

where it is reconverted to glucose (Section 23-1C).

Contrary to the widely held belief, it is not lactate

buildup in the muscle per se that causes muscle fatigue and

soreness but the accumulation of glycolytically generated

acid (muscles can maintain their workload in the presence

of high lactate concentrations if the pH is kept constant;

but see Section 27-2B). Indeed, it is well known among

hunters that the meat of an animal that has run to exhaus-

tion before being killed has a sour taste. This is a result of

lactic acid buildup in the muscles.

2 lactate ⫹ 2ATP ⫹ 2H

O ⫹ 2H

⫹

Glucose ⫹ 2ADP ⫹ 2P

Section 17-3. Fermentation: The Anaerobic Fate of Pyruvate 615

Figure 17-23 The active site region of porcine H

LDH in

complex with S-lac-NAD

ⴙ

, a covalent adduct of lactate and

NAD

ⴙ

. The adduct is shown in ball-and-stick form with C green,

N blue, O red, and P yellow, except for the covalent bond

between the methylene substituent to lactate atom C3 and

nicotinamide atom C5, which is light green. The three LDH side

chains that form hydrogen bonds (white lines) with the pyruvate

residue are shown in stick form with C magenta and N blue.

[Based on an X-ray structure by Michael Rossmann, Purdue

University. PDBid 5LDH.]

Figure 17-24 Reaction mechanism of lactate dehydrogenase.

The reaction involves direct hydride transfer from NADH to

pyruvate’s carbonyl carbon atom accompanied by proton

donation from the imidazolium group of His 195 to the pyruvate

carbonyl oxygen atom.The latter process is facilitated by the

positive charge on the nearby side chain of Arg 109.

. . .

...

–

Arg 171

Arg 109

His 195

PyruvateNADH

NAD

–

L-Lactate

JWCL281_c17_593-637.qxd 6/10/10 1:06 PM Page 615

B. Alcoholic Fermentation

Under anaerobic conditions in yeast, NAD



is regenerated

in a manner that has been of importance to mankind for

thousands of years: the conversion of pyruvate to ethanol

and CO

. Ethanol is, of course, the active ingredient of wine

and spirits; CO

so produced leavens bread. From the point

of view of the yeast, however, alcoholic fermentation has a

practical benefit that homolactic fermentation cannot sup-

ply: Yeast employ ethanol as a kind of antibiotic to elimi-

nate competing organisms. This is because yeast can grow

in ethanol concentrations 12% (2.5M), whereas few

other organisms can survive in 5% ethanol (recall that

ethanol is a widely used antiseptic).

TPP Is an Essential Cofactor of Pyruvate

Decarboxylase

Yeast produces ethanol and CO

via two consecutive re-

actions (Fig. 17-25). The first reaction is the decarboxyla-

tion of pyruvate to form acetaldehyde and CO

as cat-

alyzed by pyruvate decarboxylase (PDC; an enzyme not

present in animals). PDC contains the coenzyme thiamine

pyrophosphate [TPP; Fig. 17-26; also called thiamin

diphosphate (ThDP)], which it binds tightly but noncova-

lently.The coenzyme is employed because decarboxylation

of an -keto acid such as pyruvate requires the buildup of

negative charge on the carbonyl carbon atom in the transi-

tion state, an unstable situation:

–

This transition state may be stabilized by delocalization of

the developing negative charge into a suitable “electron

sink.” The amino acid residues of proteins function poorly

in this capacity but TPP does so easily.

The “business” end of TPP is the thiazolium ring (Fig.

17-26). Its group is relatively acidic because of the

adjacent positively charged quaternary nitrogen atom,

which electrostatically stabilizes the carbanion formed on

dissociation of the proton. This dipolar carbanion (or ylid)

is the active form of the coenzyme.The mechanism of PDC

catalysis is as follows (Fig. 17-27):

Step 1 Nucleophilic attack by the ylid form of TPP on

the carbonyl carbon of pyruvate to form a covalent adduct.

Step 2 Departure of CO

to generate a resonance-

stabilized carbanion adduct in which the thiazolium ring of

the coenzyme acts as an electron sink.

Step 3 Protonation of the carbanion.

Step 4 Elimination of the TPP ylid to form acetalde-

hyde and regenerate the active enzyme.

This mechanism has been corroborated by the isolation of

the hydroxyethylthiamine pyrophosphate intermediate

(Fig. 17-27).

The X-ray structure of PDC in complex with TPP (Fig.

17-28),which was determined by William Furey and Martin

Sax, has suggested a role for TPP’s aminopyrimidine ring in

the formation of the active ylid. Ylid formation requires a

base to remove the C2 proton. Yet PDC has no basic side

chain that is properly positioned to do so.The amino group

of the enzyme-bound TPP’s aminopyrimidine ring is suit-

ably positioned to accept this proton; however,its pK is too

low to do so efficiently and one of its protons sterically

C2¬H

616 Chapter 17. Glycolysis

Figure 17-25 The two reactions of alcoholic fermentation. (1) Decarboxylation of pyruvate to

form acetaldehyde is followed by (2) reduction of the acetaldehyde to ethanol by NADH.

Figure 17-26 Thiamine pyrophosphate. The thiazolium ring constitutes its catalytically active

functional group.

Pyruvate

pyruvate

decarboxylase

CCCH

–

Acetaldehyde

CCH

Ethanol

alcohol

dehydrogenase

NAD

NADH

PPO

–

1

2

3

4

5

6

acidic

proton

Thiazolium

ring

Aminopyrimidine

ring

JWCL281_c17_593-637.qxd 2/26/10 1:38 PM Page 616

clashes with the C2 proton. It is therefore proposed that

the aminopyrimidine is converted to its imino tautomeric

form on the enzyme’s surface in a reaction involving pro-

ton donation by Glu 51 (Fig. 17-29). The imine, in turn, ac-

cepts a proton from C2, thereby forming the ylid, with tau-

tomerization back to the amino form. The participation of

N1¿ and the 4¿-amino group of the aminopyrimidine is sup-

ported by experiments showing that TPP analogs missing

either of these functionalities are catalytically inactive.

H/D exchange experiments followed by

H NMR analysis

of the exchange products indicate that when TPP is bound

to PDC in complex with the substrate analog pyruvamide

(CH

¬CO¬CO¬NH

), its rate of exchange to form the

active species (ylid) is much greater (⬎6 ⫻ 10

⫺1

) than

the enzyme’s catalytic rate (k

cat

⫽ 10 s

⫺1

). Moreover, the

mutation of PDC’s Glu 51 to Gln reduces the rate of H/D

exchange to 1.7 s

⫺1

, thereby supporting Glu 51’s postulated

function of donating a proton to N1¿ of TPP’s aminopyri-

dine ring.

b. Beriberi Is a Thiamine Deficiency Disease

The ability of TPP’s thiazolium ring to add to carbonyl

groups and act as an “electron sink” makes it the coenzyme

most utilized in ␣-keto acid decarboxylations. TPP is also

Section 17-3. Fermentation: The Anaerobic Fate of Pyruvate 617

Figure 17-27 Reaction mechanism of pyruvate decarboxylase.

(1) Nucleophilic attack by the ylid form of TPP on the carbonyl

carbon of pyruvate; (2) departure of CO

to generate a

Figure 17-28 A portion of the X-ray structure of pyruvate

decarboxylase from Saccharomyces uvarum (brewer’s yeast) in

complex with its TPP cofactor. The enzyme’s identical

563-residue subunits form a tightly associated dimer, two of

which associate loosely to form a tetramer.The TPP and the side

chain of Glu 51 are shown in stick form with C green, N blue, O

red, S yellow, and P gold. The TPP binds in a cavity situated

between the dimer’s two subunits (cyan and magenta), where it

hydrogen bonds to Glu 51. [Based on an X-ray structure by

William Furey and Martin Sax, Veterans Administration Medical

Center and University of Pittsburgh, Pittsburgh, Pennsylvania.

PDBid 1PYD.]

See Interactive Exercise 9

C O

–

R⬘

–

R⬘

Pyruvate TPP (ylid form) TPP

–

CHO

R⬘

Hydroxyethylthiamine

pyrophosphate

H C

cetaldehyde

R⬘

–

Resonance-stabilized carbanion

resonance-stabilized carbanion; (3) protonation of the carbanion;

and (4) elimination of the TPP ylid and release of product.

JWCL281_c17_593-637.qxd 10/19/10 7:26 AM Page 617

involved in decarboxylation reactions that we shall encounter

in other metabolic pathways. Consequently, thiamine (vita-

min B

), which is neither synthesized nor stored in signifi-

cant amounts by the tissues of most vertebrates, is required

in their diets. Its deficiency in humans results in an ultimately

fatal condition known as beriberi (Sinhalese for weakness)

that is characterized by neurological disturbances causing

pain, paralysis and atrophy (wasting) of the limbs, and/or

cardiac failure resulting in edema (the accumulation of fluid

in tissues and body cavities).Beriberi was particularly preva-

lent in the late nineteenth and early twentieth centuries in

the rice-consuming areas of Asia after the introduction of

steam-powered milling machines that polished the rice

grains to remove their coarse but thiamine-containing outer

layers (the previously used milling procedures were less effi-

cient and hence left sufficient thiamine on the grains). Par-

boiling rice before milling, a process common in India,

causes the rice kernels to absorb nutrients from their outer

layers, thereby decreasing the incidence of beriberi. Once

thiamine deficiency was recognized as the cause of beriberi,

enrichment procedures were instituted so that today it has

ceased to be a problem except in areas undergoing famine.

However, beriberi occasionally develops in chronic alco-

holics due to their penchant for drinking but not eating.

c. Reduction of Acetaldehyde

and Regeneration of NAD

ⴙ

The acetaldehyde formed by the decarboxylation of

pyruvate is reduced to ethanol by NADH in a reaction cat-

alyzed by alcohol dehydrogenase (ADH). Each subunit of

the tetrameric yeast ADH (YADH) binds one NADH and

one Zn

2⫹

ion. The Zn

2⫹

ion functions to polarize the car-

bonyl group of acetaldehyde (Fig. 17-30), so as to stabilize

the developing negative charge in the transition state of the

reaction (the role of metal ions in enzymes is discussed in

Section 15-1C). This facilitates the transfer of NADH’s

pro-R hydrogen (the same atom that LDH transfers) to

acetaldehyde’s re face, forming ethanol with the transferred

hydrogen in the pro-R position (Section 13-2A).

Both homolactic and alcoholic fermentation have the

same function: the anaerobic regeneration of NAD

⫹

for

continued glycolysis. Their main difference is in their meta-

bolic products.

Mammalian liver ADH (LADH) functions to metabo-

lize the alcohols anaerobically produced by intestinal flora

as well as those from external sources (the direction of the

ADH reaction varies with the relative concentrations of

618 Chapter 17. Glycolysis

⫹

Glu 51

⫺

Glu 51

3⬘

6⬘

4⬘

5⬘

1⬘

2⬘

⫺

⫹

⫺

Ylid

Imine Predominant form

fast

Steric clash

⫹

⫺

⬘

6⬘

4⬘

5⬘

1⬘

2⬘

Glu 51

⫹

⫺

⬘

6⬘

4⬘

5⬘

1⬘

2⬘

Figure 17-29 The formation of the active ylid form of TPP in

the pyruvate decarboxylase reaction. This reaction requires the

participation of TPP’s aminopyrimidine ring together with

general acid catalysis by Glu 51.The predominant form of the

cofactor on the enzyme is the imine, but the rate of formation of

the active ylid is fast relative to the enzyme’s catalytic rate.

Ethanol

Acetaldehyde

NADH

NAD

...

S-Cys

N-His

C OH

Figure 17-30 The reaction mechanism of alcohol

dehydrogenase involves direct hydride transfer of the pro-R

hydrogen of NADH to the re face of acetaldehyde.

JWCL281_c17_593-637.qxd 6/3/10 8:37 AM Page 618

ethanol and acetaldehyde). Each subunit of this dimeric

enzyme binds one NAD

⫹

and two Zn

2⫹

ions, although only

one of these ions participates directly in catalysis. There is

significant amino acid sequence similarity between YADH

and LADH, so it is quite likely that both enzymes have the

same general mechanism.

C. Energetics of Fermentation

Thermodynamics permits us to dissect the process of fer-

mentation into its component parts and to account for the

free energy changes that occur.This enables us to calculate

the efficiency with which the free energy of degradation of

glucose is utilized in the synthesis of ATP. The overall reac-

tion of homolactic fermentation is

(⌬G°¿ is calculated from the data in Table 3-4 using Eqs.

[3.19] and [3.21] adapted for 2H

⫹

ions.) For alcoholic fer-

mentation, the overall reaction is

Each of these reactions is coupled to the net formation of

two ATPs, which requires ⌬G°¿ ⫽⫹61 kJ ⴢ mol

⫺1

of glucose

consumed (Table 16-3). Dividing the ⌬G°¿ of ATP forma-

tion by that of lactate formation indicates that homolactic

fermentation is 31% “efficient”; that is, 31% of the free en-

ergy released by this process under standard biochemical

conditions is sequestered in the form of ATP. The rest is

dissipated as heat, thereby making the process irreversible.

Likewise, alcoholic fermentation is 26% efficient under

biochemical standard state conditions. Actually, under

physiological conditions, where the concentrations of reac-

tants and products differ from those of the standard state,

these reactions have free energy efficiencies of ⬎50%.

a. Glycolysis Is Used for Rapid ATP Production

Anaerobic fermentation utilizes glucose in a profligate

manner compared to oxidative phosphorylation: Fermenta-

tion results in the production of 2 ATPs per glucose, whereas

oxidative phosphorylation yields 32 ATPs per glucose

(Chapter 22). This accounts for Pasteur’s observation that

yeast consumes far more sugar when growing anaerobically

than when growing aerobically (the Pasteur effect; Section

22-4C). However, the rate of ATP production by anaerobic

glycolysis can be up to 100 times faster than that of oxidative

phosphorylation. Consequently, when tissues such as muscle

are rapidly consuming ATP, they regenerate it almost entirely

by anaerobic glycolysis. (Homolactic fermentation does not

really “waste” glucose since the lactate so produced is aero-

bically reconverted to glucose by the liver; Section 23-1C).

Skeletal muscles consist of both slow-twitch (Type I) and

fast-twitch (Type II) fibers. Fast-twitch fibers, so called be-

cause they predominate in muscles capable of short bursts

of rapid activity, are nearly devoid of mitochondria, so that

they must obtain nearly all of their ATP through anaerobic

¢G°¿ ⫽⫺235 kJ ⴢ mol

⫺1

of glucose

Glucose

2CO

⫹ 2 ethanol

¢G°¿ ⫽⫺196 kJ ⴢ mol

⫺1

of glucose

Glucose

2 lactate ⫹ 2H

⫹

glycolysis, for which they have a particularly large capacity.

Muscles designed to contract slowly and steadily, in contrast,

are enriched in slow-twitch fibers that are rich in mitochon-

dria and obtain most of their ATP through oxidative phos-

phorylation. (Fast- and slow-twitch fibers were originally

known as white and red fibers, respectively, because other-

wise pale colored muscle tissue, when enriched with mito-

chondria, takes on the red color characteristic of their heme-

containing cytochromes. However, fiber color has been

shown to be an imperfect indicator of muscle physiology.)

In a familiar example, the flight muscles of migratory

birds such as ducks and geese, which need a continuous en-

ergy supply, are rich in slow-twitch fibers and therefore

such birds have dark breast meat. In contrast, the flight

muscles of less ambitious fliers, such as chickens and

turkeys, which are used only for short bursts (often to es-

cape danger), consist mainly of fast-twitch fibers that form

white meat. In humans, the muscles of sprinters are rela-

tively rich in fast-twitch fibers, whereas distance runners

have a greater proportion of slow-twitch fibers (although

their muscles have the same color). World class distance

runners have a remarkably high capacity to generate ATP

aerobically. This was demonstrated by the noninvasive

NMR monitoring of the ATP, P

, phosphocreatine, and pH

levels in their exercising but untrained forearm muscles.

These observations suggest that the muscles of these ath-

letes are better endowed genetically for endurance exer-

cise than those of “normal” individuals.

4 METABOLIC REGULATION

AND CONTROL

Living organisms, as we saw in Section 16-6, are thermody-

namically open systems that tend to maintain a steady state

rather than reaching equilibrium (death for living things).

Thus the flux (rate of flow) of intermediates through a meta-

bolic pathway is constant; that is, the rates of synthesis and

breakdown of each pathway intermediate maintain it at a

constant concentration. Such a state, it will be recalled, is

one of maximum thermodynamic efficiency (Section 16-6Ba).

Regulation of the steady state (homeostasis) must be main-

tained in the face of changes in flux through the pathway in

response to changes in demand.

The terms metabolic control and metabolic regulation

are often used interchangeably. However, for our purposes

we shall give them different definitions: Metabolic regula-

tion is the process by which the steady-state flow of

metabolites through a pathway is maintained, whereas

metabolic control is the influence exerted on the enzymes

of a pathway in response to an external signal in order to

alter the flux of metabolites.

A. Homeostasis and Metabolic Control

There are two reasons why metabolic flow must be con-

trolled:

I. To provide products at the rate they are needed, that

is, to balance supply with demand.

Section 17-4. Metabolic Regulation and Control 619

JWCL281_c17_593-637.qxd 6/3/10 8:37 AM Page 619

II. To maintain the steady-state concentrations of the

intermediates in a pathway within a narrow range (home-

ostasis).

Organisms maintain homeostasis for several reasons:

1. In an open system, such as metabolism, the steady

state is the state of maximum thermodynamic efficiency

(Section 16-6Ba).

2. Many intermediates participate in more than one

pathway, so that changing their concentrations may disturb

a delicate balance.

3. The rate at which a pathway can respond to a control

signal slows if large changes in intermediate concentrations

are involved.

4. Large changes in intermediate concentrations may

have deleterious effects on cellular osmotic properties.

The concentrations of intermediates and the level of

metabolic flux at which a pathway is maintained vary with

the needs of the organism through a highly responsive

system of precise controls. Such pathways are analogous

to rivers that have been dammed to provide a means of

generating electricity. Although water is continually flow-

ing in and out of the lake formed by the dam, a relatively

constant water level is maintained. The rate of water out-

flow from the lake is precisely controlled at the dam and

is varied in response to the need for electrical power. In

this section, we examine the mechanisms by which meta-

bolic pathways in general, and the glycolytic pathway in

particular, are controlled in response to biological energy

needs.

B. Metabolic Flux

Since a metabolic pathway is a series of enzyme-catalyzed

reactions, it is easiest to describe the flux of metabolites

through the pathway by considering its reaction steps indi-

vidually. The flux of metabolites, J, through each reaction

step is the rate of the forward reaction, v

, less that of the

reverse reaction, v

[17.1]

At equilibrium, by definition, there is no flux (J  0), al-

though v

and v

may be quite large. At the other extreme,

in reactions that are far from equilibrium, v

 v

, so that

the flux is essentially equal to the rate of the forward reac-

tion, J ⬇ v

. The flux throughout a steady-state pathway is

constant and is set (generated) by the pathway’s rate-

determining step (or steps). Consequently, control of flux

through a metabolic pathway requires: (1) that the flux

through this flux-generating step vary in response to the or-

ganism’s metabolic requirements and (2) that this change in

flux be communicated throughout the pathway to maintain

a steady state.

The classic description of metabolic control and regula-

tion is that every metabolic pathway has a rate-limiting

step and is regulated by controlling the rate of this pivotal

J  v

 v

enzyme. These so-called regulatory enzymes are almost in-

variably allosteric enzymes subject to feedback inhibition

(Section 13-4) and are often also controlled by covalent

modification (which we discuss in Section 18-3).

Several questions arise. Are these regulatory enzymes

really rate limiting for the pathway? Is there really only

one step in the pathway that is rate limiting, or might there

be a number of enzymes contributing to the regulation of

the pathway? Does controlling these enzymes really con-

trol the flux of metabolites through the pathway or is the

function of feedback inhibition really to maintain a steady

state? These are complicated questions with complicated

answers.

C. Metabolic Control Analysis

While it has been common practice to assume that every

metabolic pathway has a rate-limiting step, experiments

suggest that the situation becomes more complex when

these pathways are combined in a living organism. Hence,

it is important to develop methods to quantitatively ana-

lyze metabolic systems in order to establish mechanisms of

control and regulation. Metabolic control analysis, devel-

oped by Henrik Kacser and Jim Burns and independently

by Reinhart Heinrich and Tom Rapoport, provides a

framework for considering these problems. It is a way of

quantitatively describing the behavior of metabolic sys-

tems in response to various perturbations.

a. The Flux Control Coefficient Measures the

Sensitivity of the Flux to the Change

in Enzyme Concentration

Metabolic control analysis makes no a priori assump-

tion that only one step is rate limiting. Instead, it defines a

flux control coefficient, C

(where J is an index, not an ex-

ponent), to measure the sensitivity of flux to a change in

enzyme concentration. The flux control coefficient is de-

fined as the fractional change in flux, J, with respect to the

fractional change in enzyme concentration, [E]:

[17.2]

(recall that 0x/x  0 ln x).

The flux control coefficient is the analog of the kinetic or-

der of a reaction. If a reaction is first order in substrate con-

centration, [S], then doubling [S] doubles the rate of the re-

action, whereas if the reaction is zero order in [S] (e.g., in a

saturated enzymatic reaction), then the reaction rate is in-

sensitive to the value of [S]. Similarly, if the flux control coef-

ficient of an enzyme is 1, then doubling the concentration of

the enzyme, [E], doubles the flux through the pathway and if

it is zero, the flux is insensitive to the value of [E]. Of course,

the flux control coefficient may have some intermediate

value between 0 and 1. For example, if a 10% increase in the

enzyme concentration increases the flux by only 7.5%, the

flux control coefficient would be 0.075/0.10  0.75.

The flux through a metabolic system is generally con-

trolled by more than one enzyme. Consequently, the flux



0J>J

0[E]>[E]



0 ln J

0 ln[E]

⬇

¢J>J

¢[E]>[E]

620 Chapter 17. Glycolysis

JWCL281_c17_593-637.qxd 2/26/10 1:38 PM Page 620