Voet D., Voet Ju.G. Biochemistry

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–

OPO

–

Enzyme

–

...

OPO

–

Enzyme

Are the two classes of aldolases related? Although both

classes exhibit the Uni Bi kinetics implicit in their mecha-

nisms, they exhibit only ⬃15% sequence identity, which

places them in the lower end of the twilight zone for estab-

lishing homology (Section 7-4Ba). Nevertheless, their X-ray

structures reveal that they have the same fold, the /

barrel. The evolution of this particularly common fold is

discussed in Section 8-3Bh.

b. Why Two Classes of Aldolase?

Since glycolysis presumably arose very early in evolu-

tionary history, the existence of two classes of aldolase is

unexpected. It had originally been postulated that, since

Class I aldolases occur in higher organisms, Class II al-

dolases must be the more primitive enzyme form, that is,

less metabolically capable than are the Class I enzymes.

However, the discovery that some organisms simultane-

ously express both classes of aldolase suggests that both

enzyme classes are evolutionarily ancient and equally

interconvertible C3 compounds that can therefore enter a

common degradative pathway.The enolate intermediate in

the aldol cleavage reaction is stabilized by resonance, as

shown, as a result of the electron-withdrawing character of

the carbonyl oxygen atom.

Note that at this point in the pathway the atom number-

ing system changes. Atoms 1, 2, and 3 of glucose become

atoms 3, 2, and 1 of DHAP, thus reversing order. Atoms 4,

5, and 6 become atoms 1, 2, and 3 of GAP (Fig. 17-3).

a. There Are Two Mechanistic Classes of Aldolases

Aldol cleavage is catalyzed by stabilizing its enolate in-

termediate through increased electron delocalization.

There are two types of aldolases that are classified accord-

ing to the chemistry they employ to stabilize the enolate. In

Class I aldolases, which occur in animals and plants, the re-

action occurs as follows (Fig. 17-9):

Step 1 Substrate binding.

Step 2 Reaction of the FBP carbonyl group with the

e-amino group of the active site Lys 229 to form an

iminium cation, that is, a protonated Schiff base.

Step 3 bond cleavage resulting in enamine for-

mation and the release of GAP. The iminium ion, as we saw

in Section 16-2E, is a better electron-withdrawing group

than is the oxygen atom of the precursor carbonyl group.

Thus, catalysis occurs because the enamine intermediate

(Fig. 17-9, Step 3) is more stable than the corresponding

enolate intermediate of the base-catalyzed aldol cleavage

reaction (Fig. 17-8, Step 2).

Step 4 Protonation of the enamine to an iminium cation.

Step 5 Hydrolysis of this iminium cation to release

DHAP, with regeneration of the free enzyme.

Proof for the formation of the Schiff base in Step 2 was

provided by “trapping”

C-labeled DHAP on the enzyme

by reacting it with NaBH

, which reduces imines to

amines:

The radioactive product was hydrolyzed and identified as

-␤-glyceryl lysine.

OPO

2

NaBH

reduction

(CH

)

NH Enzyme

hydrolysis



(CH

)

COO



--Glyceryl lysine



C3¬O4

Cys and His residues were initially thought to act as the

acid and base catalysts that facilitate the proton transfers in

the aldolase reaction because the appropriate group-

specific reagents inactivate the enzyme by reacting with

these residues. For example, the reaction of a specific Cys

residue of aldolase with iodoacetic acid inactivates the en-

zyme and results in the buildup of the FBP observed in the

early glycolysis inhibition studies (Section 17-1A). How-

ever, site-directed mutagenesis to Ala of the Cys residue

thought to be involved in the catalytic activity results in no

loss of enzymatic function. Modification of this Cys residue

apparently prevents the conformational changes required

for productive substrate binding.

An early X-ray structure of aldolase suggested that a

Tyr side chain was positioned to act as the active site

acid–base catalyst and that the His was instead necessary

for the maintenance of the Tyr’s catalytically active orien-

tation. A re-examination of the X-ray data caused yet an-

other modification of the mechanism. The Tyr originally

seen at the active site has changed position in this new

analysis, so that it is out of reach of the active site. Asp 33

and Lys 229 now appear to be acting as acid–base catalysts.

These residues are evolutionarily conserved and their mu-

tagenesis eliminates enzyme activity. This is an excellent

example of the caution that must be exercised in the inter-

pretation of chemical modification and structural data, and

the power of site-directed mutagenesis in the study of en-

zyme mechanisms (although see Section 15-3Ba).

Class II aldolases, which occur in fungi, algae, and some

bacteria, do not form a Schiff base with the substrate.

Rather, a divalent cation, usually Zn

2

or Fe

2

, polarizes

the carbonyl oxygen of the substrate to stabilize the eno-

late intermediate of the reaction (Fig. 16-12d):

Section 17-2. The Reactions of Glycolysis 601

JWCL281_c17_593-637.qxd 6/30/10 10:53 AM Page 601

602 Chapter 17. Glycolysis

–

OPO

–

(CH

)

OCH

Fructose-1,6-bisphosphate

Lys 229

(CH

)

OCH

. . .

substrate

binding

(CH

)

Enamine

intermediate

. . .

H OH

Glyceraldehyde-

3-phosphate

(product 1)

(CH

)

Enzyme –product

protonated Schiff base

pro–R

pro–S

(CH

)

Dihydroxyacetone

phosphate

(product 2)

OPO

–

OPO

–

OPO

–

OPO

–

OPO

–

OPO

–

OPO

–

. . .

–

Schiff base

hydrolysis

Free enzyme

Asp 33

Enzyme –substrate

complex

protonated Schiff

base formation

Enzyme–substrate

protonated Schiff base

tautomerization

protonation

aldol cleavage

Figure 17-9 Enzymatic mechanism of Class I aldolase. The

reaction involves (1) substrate binding; (2) Schiff base formation

between the enzyme’s active site Lys residue and FBP; (3) aldol

cleavage to form an enamine intermediate of the enzyme and

DHAP with release of GAP (shown with its re face up);

(4) tautomerization and protonation to the iminium form of the

Schiff base; and (5) hydrolysis of the Schiff base with release of

DHAP.

See the Animated Figures.

JWCL281_c17_593-637.qxd 2/26/10 1:37 PM Page 602

adept at carrying out their metabolic functions. Thus, the

expression of both classes of aldolase in some organisms

probably represents an ancient metabolic redundancy that

evolution has eliminated in most contemporary organisms.

Whatever the reason for the occurrence of two classes of

aldolase, the fact that Class II aldolases do not occur in

mammals makes them an attractive target in the develop-

ment of antibacterial drugs.

c. Aldolase Is Stereospecific

The aldolase reaction provides another example of the

extraordinary stereospecificity of enzymes. In the nonenzy-

matic aldol condensation to form hexose-1,6-bisphosphate

from DHAP and GAP, there are four possible products de-

pending on whether the pro-R or pro-S hydrogen at C3 of

DHAP is removed and whether the resulting carbanion

attacks GAP on its re or its si face:

In the enzymatic aldol condensation (Fig. 17-9 in reverse),

carbanion formation from the enzyme–DHAP iminium ion

(Fig.17-9, Step 4 in reverse) occurs with removal of only the

pro-S hydrogen. Attack of this carbanion occurs only on

the si face of the enzyme-bound GAP carbonyl group, so

that only FBP is formed (Fig. 17-9, Step 3 in reverse).

E. Triose Phosphate Isomerase

Only one of the products of the aldol cleavage reaction,

GAP, continues along the glycolytic pathway (Fig. 17-3).

However, DHAP and GAP are ketose–aldose isomers just

as are F6P and G6P. Interconversion of GAP and DHAP

therefore probably occurs via an enediol or enediolate in-

termediate in analogy with the phosphoglucose isomerase

reaction (Fig. 17-6). Triose phosphate isomerase (TIM or

OPO

2

OPO

2

HO H

HOH

OPO

2

OPO

2

HOH

D-Fructose

1,6-bisphosphate

D-Psicose

1,6-bisphosphate

OPO

2

OPO

2

HO H

OPO

2

OPO

2

HO H

D-Tagatose

1,6-bisphosphate

D-Sorbose

1,6-bisphosphate

TPI; Figs. 8-19b and 8-52) catalyzes this process in Reac-

tion 5 of glycolysis, the final reaction of Stage I:

Support for this reaction scheme comes from the use of

the transition state analogs phosphoglycohydroxamate and

2-phosphoglycolate, stable compounds whose geometric

structures resemble that of the proposed enediol or enedi-

olate intermediate:

Since enzymes catalyze reactions by binding the transition

state complex more tightly than the substrate (Section

15-1F), phosphoglycohydroxamate and 2-phosphoglycolate

should bind more tightly to TIM than does substrate. In

fact, phosphoglycohydroxamate and 2-phosphoglycolate

bind 155- and 100-fold more tightly to TIM than do either

GAP or DHAP.

a. Glu 165 Functions as a General Base

The pH dependence of the TIM reaction is a bell-

shaped curve with pK’s of 6.5 and 9.5. The similarity of

these pK’s to the corresponding quantities of the phospho-

glucose isomerase reaction suggests the participation of

both an acid and a base in the TIM reaction as well. How-

ever, pH studies alone, as we have seen, are difficult to

–

Phosphoglyco-

hydroxamate

OPO

–

OPO

–

OPO

–

2-Phosphoglycolate

–

Proposed enediolate

intermediate

Glyceraldehyde-

3-phosphate

(an aldose)

Enediol

intermediate

Dihydroxyacetone

phosphate

(a ketose)

OHH

COH

OHH

OPO

–

OPO

–

OPO

–

Section 17-2. The Reactions of Glycolysis 603

JWCL281_c17_593-637.qxd 2/26/10 1:37 PM Page 603

interpret in terms of specific amino acid residues since the

active site environment may alter the pK of an acidic or ba-

sic group.

Affinity labeling reagents have been employed in an ef-

fort to identify the base at the active site of TIM. Both bro-

mohydroxyacetone phosphate and glycidol phosphate

inactivate TIM by forming esters of Glu 165, whose car-

boxylate group, X-ray studies indicate, is ideally situated to

abstract the C2 proton from the substrate (general base

catalysis). In fact, the mutagenic replacement of Glu 165 by

Asp, which X-ray studies show withdraws the carboxylate

group only ⬃1 Å farther away from the substrate than its

position in the wild-type enzyme, reduces TIM’s catalytic

power ⬃1000-fold. Note that Glu 165’s pK is drastically al-

tered from the 4.1 value of the free amino acid to the ob-

served 6.5 value. This provides yet another striking exam-

ple of the effect of environment on the properties of amino

acid side chains.

b. The TIM Reaction Probably Occurs via Concerted

General Acid–Base Catalysis Involving Low-Barrier

Hydrogen Bonds

The X-ray structure of yeast TIM in complex with

phosphoglycohydroxamate indicates that His 95 is hydro-

gen bonded to and hence is properly positioned to proto-

nate the carbonyl oxygen atom of GAP (general acid

catalysis):

However, NMR studies indicate that His 95 is in its neutral

imidazole form rather than its protonated imidazolium

form. How can an imidazole group, which has a

highly basic pK of ⬃14, protonate a carbonyl oxygen atom

N3¬H

Glu 165

Asn 10

His 95

Ser 96

Glu 97





Lys 12

2

OPO

2

OPO

2

Bromohydroxyacetone

phosphate

Glycidol phosphate

that, when protonated, has a very acidic pK of 0? Like-

wise, how can the Glu 165 carboxylate group (pK 6.5) ab-

stract the C2 proton from GAP (pK ⬃17)? A plausible an-

swer is that these proton shifts are facilitated by the

formation of low-barrier hydrogen bonds (LBHBs). These

unusually strong associations (40 to 80 kJ ⴢ mol

1

12 to 30 kJ ⴢ mol

1

for normal hydrogen bonds), as we

have seen in the case of the serine protease catalytic triad

(Section 15-3Dd), form when the pK’s of the hydrogen

bonding donor and acceptor groups are nearly equal.They

can be important contributors to rate enhancement if they

form only in the transition state of an enzymatically cat-

alyzed reaction.

In converting GAP to the enediol (or enediolate) inter-

mediate (Fig. 17-10, left), the pK of the protonated form of

its carbonyl oxygen, which becomes a hydroxyl group, in-

creases to ⬃14, which closely matches that of neutral His

95. The resulting LBHB between this hydroxyl group and

His 95 permits the neutral imidazole side chain to proto-

nate the oxygen atom. Likewise, as the carbonyl oxygen is

protonated, the pK of the proton decreases to ⬃7,

close to the pK of the Glu 165 carboxylate. It therefore ap-

pears that the reaction occurs via simultaneous proton ab-

straction by Glu 165 and protonation by His 95 (concerted

general acid–base catalysis). The LBHBs postulated to

form in the transition state, but not in the Michaelis com-

plex, between Glu 165 and C2¬H and between His 95 and

the carbonyl oxygen atom are thought to provide some of

the transition state stabilization necessary to catalyze the

reaction.The positively charged side chain of Lys 12, which

is probably responsible for the 9.5 pK observed in TIM’s

pH rate profile, is thought to electrostatically stabilize the

negatively charged transition state. The conversion of the

enediol(ate) intermediate to DHAP is likewise facilitated

by the formation of transition state LBHBs (Fig. 17-10,

right). In fact, in the high resolution (1.2 Å) X-ray structure

of TIM in complex with DHAP, determined by Ann

McDermott and Liang Tong, the hydrogen bond between

His 95 and O2 of DHAP has an unusually short distance of

2.6 Å. In addition, a carboxylate oxygen atom of Glu 165

forms extraordinarily close contacts of ⬃3.0 Å with both

C1 and C2 of DHAP.

c. A Flexible Loop Both Preferentially Binds

and Protects the Enediol Intermediate

The comparison of the X-ray structure of the TIM ⴢ

phosphoglycohydroxamate complex with that of TIM

alone reveals that a 10-residue loop, which is closed over

the active site in the enzyme–substrate complex, is flipped

up in the unoccupied active site like a hinged lid, a move-

ment that involves main chain shifts of 7 Å (Fig. 17-11).A

four-residue segment of this loop makes a hydrogen bond

with the phosphate group of the substrate. Mutagenic exci-

sion of these four residues neither significantly distorts the

protein nor greatly impairs substrate binding.The catalytic

power of the mutant enzyme is, nevertheless, reduced 10

fold and it only weakly binds phosphoglycohydroxamate.

Evidently, the closed loop preferentially stabilizes the en-

zymatic reaction’s enediol-like transition state.

C2¬H

604 Chapter 17. Glycolysis

JWCL281_c17_593-637.qxd 2/26/10 1:37 PM Page 604

Section 17-2. The Reactions of Glycolysis 605

Figure 17-10 Proposed enzymatic mechanism of the TIM

reaction. The reaction proceeds via the concerted abstraction of

the proton of GAP by the carboxylate group of Glu 165

and the protonation of the GAP carbonyl oxygen atom by the

imidazole group of His 95.The pK’s of the corresponding donor

and acceptor groups participating in each proton transfer process

become nearly equal in the transition state and hence are

C2¬H

Figure 17-11 Ribbon diagram of yeast TIM in complex with its

transition state analog 2-phosphoglycolate. A single 248-residue

subunit of this homodimeric enzyme is viewed roughly along the

axis of its ␣/␤ barrel.The enzyme’s flexible loop, residues 168

through 177, is cyan and the side chains of Lys 12, His 95, and

Glu 165 are purple, magenta, and red, respectively. The

2-phosphoglycolate is shown in space-filling form colored

according to atom type (C green, O red, P yellow). [Based on an

X-ray structure by Gregory Petsko, Brandeis University. PDBid

2YPI.]

See Interactive Exercise 2 and Kinemage Exercises

12-1 and 12-2

OPO

⫺

GAP

•

TIM Michaelis complex

DHAP

•

TIM Michaelis complex

Transition state

⫺

Glu

165

His

OPO

⫺

Glu

165

His

195

OPO

⫺

Glu

165

His

OPO

⫺

Glu

165

His

Enediol (or enendiolate) intermediate

⫺

OPO

⫺

Glu

165

His

proposed to form low-barrier hydrogen bonds (red dashed lines),

which act to stabilize the transition state. The resulting enediol

(or possibly the electrostatically stabilized enediolate) intermedi-

ate then reacts in a similar fashion with the carboxyl group of

Glu 165 protonating C1, while the deprotonated N3 atom of His

95 abstracts the proton on the 2-hydroxyl group to yield DHAP.

JWCL281_c17_593-637.qxd 10/19/10 7:26 AM Page 605

The closed loop conformation in the TIM reaction pro-

vides a striking example of the stereoelectronic control that

enzymes can exert on a reaction (Section 15-1Eb). In solu-

tion, the enediol intermediate readily breaks down with the

elimination of the phosphate at C3 to form the toxic com-

pound methylglyoxal (Fig. 17-12a). On the enzyme’s surface,

however, this reaction is prevented because the phosphate

group is held by the flexible loop in the plane of the enediol,

a position that disfavors phosphate elimination. In order for

this elimination to occur, the bond to the phosphate

group must lie, as shown in Fig. 17-12a, in the plane perpen-

dicular to that of the enediol. This is because, if the phos-

phate group were to be eliminated while this bond

was in the plane of the enediol as diagrammed in Fig. 17-

12b, the CH

group of the resulting enol product would be

twisted 90° out of the plane of the rest of the molecule. Such

a conformation is energetically prohibitive because it pre-

vents the formation of the enol’s double bond by eliminat-

ing the overlap between its component p orbitals. In the

mutant enzyme lacking the flexible loop, the enediol is able

to escape: ⬃85% of the enediol intermediate is released

into solution, where it rapidly decomposes to methylglyoxal

and P

.Thus, flexible loop closure also ensures that substrate

is efficiently transformed to product.

On the basis of the foregoing X-ray structures, it had

been widely assumed that the binding of substrate to TIM is

ligand-gated, that is, it induces loop closure. Yet, if this were

the case, the reversibility of the TIM reaction and the chem-

ical resemblance of its reactant and product (GAP and

DHAP) make it difficult to rationalize how product could

be released. However, NMR measurements by John

Williams and McDermott reveal that, in fact, loop motion

still occurs when TIM is binding either glycerol-3-phosphate

(a substrate analog) or 2-phosphoglycolate (a transition

state analog) and is sufficiently fast (on a time scale of 100

␮s) to account for the catalytic reaction rate (a turnover

C¬O

time of 230 ␮s).This is a clear example of how complemen-

tary information supplied by X-ray and NMR methods has

yielded important insights into an enzymatic mechanism

that neither technique alone could have provided.

d. TIM Is a Perfect Enzyme

TIM, as Jeremy Knowles demonstrated, has achieved

catalytic perfection in that the rate of bimolecular reaction

between enzyme and substrate is diffusion controlled; that

is, product formation occurs as rapidly as enzyme and sub-

strate can collide in solution, so that any increase in TIM’s

catalytic efficiency would not increase the reaction rate

(Section 14-2Bb). Because of the high interconversion effi-

ciency of GAP and DHAP, these two metabolites are

maintained in equilibrium: K ⫽ [GAP]/[DHAP] ⫽ 4.73 ⫻

⫺2

; that is, [DHAP] ⬎⬎ [GAP] at equilibrium. However,

as GAP is utilized in the succeeding reaction of the gly-

colytic pathway, more DHAP is converted to GAP, so that

these compounds maintain their equilibrium ratio. One

common pathway therefore accounts for the metabolism

of both products of the aldolase reaction.

Let us now take stock of where we are in our travels

down the glycolytic pathway. At this stage, the glucose,

which has been transformed into two GAPs, has com-

pleted the preparatory stage of glycolysis. This process has

required the expenditure of two ATPs. However, this in-

vestment has resulted in the conversion of one glucose to

two C

units, each of which has a phosphoryl group that,

with a little chemical artistry, can be converted to a “high-

energy” compound (Section 16-4Ba) whose free energy of

hydrolysis can be coupled to ATP synthesis. This energy

investment is doubly repaid in the final stage of glycolysis in

which the two phosphorylated C

units are transformed to

two pyruvates with the coupled synthesis of four ATPs per

glucose.

606 Chapter 17. Glycolysis

Figure 17-12 The spontaneous

decomposition of the enediol

intermediate in the TIM reaction to

form methylglyoxal through the

elimination of a phosphate group. (a)

This reaction can occur only when the

bond to the phosphate group lies

in a plane that is nearly perpendicular

to that of the enediol so as to permit

the formation of a double bond in the

intermediate enol product. (b) When

the bond to the phosphate group

lies in a plane that is nearly parallel to

that of the enediol, the p orbitals on the

resulting intermediate product would

be perpendicular to each other and

hence lack the overlap necessary to

form a ␲ bond, that is, a double bond.

The resulting unsatisfied bonding

capacity greatly increases the energy of

the reaction intermediate and hence

makes the reaction highly unfavorable.

C¬O

Parallel p orbitals

maximally

overlapped to

form a π bond

Perpendicular p orbitals

have no overlap; a π

bond cannot be formed

O bond to phosphate

in plane perpendicular

to plane of molecule

O bond to phosphate

in plane of molecule

Enol Methyl

glyoxal

Enediol

intermediate

(a)

(b)

OHC

OPO

2⫺

OHC

OPO

2⫺

OHC

JWCL281_c17_593-637.qxd 6/30/10 10:53 AM Page 606

F. Glyceraldehyde-3-Phosphate Dehydrogenase:

First “High-Energy” Intermediate Formation

Reaction 6 of glycolysis involves the oxidation and phos-

phorylation of GAP by NAD

⫹

and P

as catalyzed by

glyceraldehyde-3-phosphate dehydrogenase (GAPDH;

Figs. 8-45 and 8-53b):

OPO

–

HOH

+ NAD

+ P

Glyceraldehyde-

3-phosphate (GAP)

OPO

–

OPO

–

HOH

+ NADH

1,3-Bisphosphoglycerate

(1,3-BPG)

glyceraldehyde-3-phosphate

dehydrogenase (GAPDH)

This is the first instance of the chemical artistry alluded

to above. In this reaction, aldehyde oxidation, an exer-

gonic reaction, drives the synthesis of the acyl phosphate

1,3-bisphosphoglycerate (1,3-BPG; previously called

1,3-diphosphoglycerate). Recall that acyl phosphates are

compounds with high phosphate group-transfer potential

(Section 16-4Ba).

a. Mechanistic Studies

Several key enzymological experiments have con-

tributed to the elucidation of the GAPDH reaction mech-

anism (Fig. 17-13):

1. GAPDH is inactivated by alkylation with stoichio-

metric amounts of iodoacetate. The presence of car-

boxymethylcysteine in the hydrolysate of the resulting

alkylated enzyme (Fig. 17-13a) suggests that GAPDH has

an active site Cys sulfhydryl group.

2. GAPDH quantitatively transfers

H from C1 of

GAP to NAD

⫹

(Fig. 17-13b), thereby establishing that this

reaction occurs via direct hydride transfer.

3. GAPDH catalyzes exchange of

P between [

P]P

and the product analog acetyl phosphate (Fig. 17-13c).

Such isotope exchange reactions are indicative of an

acyl–enzyme intermediate (Section 14-5D).

Section 17-2. The Reactions of Glycolysis 607

Figure 17-13 Some reactions employed in elucidating the

enzymatic mechanism of GAPDH. (a) The reaction of

iodoacetate with an active site Cys residue. (b) Quantitative

1,3-Bisphosphoglycerate

(1,3-BPG)

(a)

Enzyme

+ ICH

COO

–

Enzyme

COO

–

COO

–

GAPDH

Active site

Cys

Iodoacetate

protein

hydrolysis

COO

–

S +

Other

amino

acids

Carboxy-

methylcysteine

(b)

+ NAD

[1-

H]GAP

OHCH

OPO

–

OPO

–

GAPDH

+ NAD

(c)

PHO

–

GAPDH

PHO

–

32 32

Acetyl phosphate

+ P

OPO

–

tritium transfer from substrate to NAD

⫹

.(c) The enzyme-

catalyzed exchange of

P from phosphate to acetyl phosphate.

JWCL281_c17_593-637.qxd 6/3/10 8:16 AM Page 607

David Trentham has proposed a mechanism for GAPDH

based on this information and the results of kinetic studies

(Fig. 17-14):

Step 1 GAP binds to the enzyme.

Step 2 The essential sulfhydryl group, acting as a nucle-

ophile, attacks the aldehyde to form a thiohemiacetal.

Step 3 The thiohemiacetal undergoes oxidation to an

acyl thioester by direct transfer of a hydride to NAD



.This

intermediate, which has been isolated, has a high group-

transfer potential. The energy of aldehyde oxidation has not

been dissipated but has been conserved through the synthe-

sis of the thioester and the reduction of NAD



to NADH.

Step 4 Another molecule of NAD



replaces NADH.

Step 5 The thioester intermediate undergoes nucle-

ophilic attack by P

to regenerate free enzyme and form

1,3-BPG. This “high-energy” mixed anhydride generates

ATP from ADP in the next reaction of glycolysis.

G. Phosphoglycerate Kinase: First ATP Generation

Reaction 7 of the glycolytic pathway results in the first for-

mation of ATP together with 3-phosphoglycerate (3PG) in

a reaction catalyzed by phosphoglycerate kinase (PGK):

(Note: The name “kinase” is given to any enzyme that trans-

fers a phosphoryl group between ATP and a metabolite.

Nothing is implied as to the exergonic direction of transfer.)

OHCH

OPO

–

OPO

–

1,3-Bisphosphoglycerate

(1,3-BPG)

+ ADP

OPO

–

OHCH

3-Phosphoglycerate

(3PG)

+ ATP

–

phosphoglycerate

kinase (PGK)

608 Chapter 17. Glycolysis

Figure 17-14 Enzymatic mechanism of glyceraldehyde-

3-phosphate dehydrogenase. (1) GAP binds to the enzyme;

(2) the active site sulfhydryl group forms a thiohemiacetal

with the substrate; (3) NAD



oxidizes the thiohemiacetal

to a thioester; (4) the newly formed NADH is replaced

on the enzyme by NAD



; and (5) P

attacks the

thioester, forming the acyl phosphate

product, 1,3-BPG, and

regenerating the

active enzyme.

See the

Animated Figures

NAD

...

Thiohemiacetal

intermediate

–

Acyl thioester

intermediate

NADH

NAD

 P

OPO

–

NAD

...

Enzyme–substrate

complex

NADH

...

–

NAD

...

NAD

...

GAP

JWCL281_c17_593-637.qxd 6/3/10 8:16 AM Page 608

The X-ray structure of yeast PGK in complex with

2⫹

–ATP and 3PG, its enzyme–product complex, was de-

termined by Herman Watson (Fig. 17-15a). Note its con-

spicuously bilobal appearance and the close approach be-

tween the ATP’s ␥-phosphate group and one of the 3PG’s

carboxylate oxygen atoms. The 3PG and the Mg–ATP are

respectively bound to PGK’s N- and C-terminal domains.

The X-ray structure of PGK from the thermophile Ther-

motoga maritima in complex with 3PG and the nonhy-

drolyzable ATP analog AMPPNP (ATP with the O atom

bridging its ␤ and ␥ phosphate groups replaced by an NH

group) reveals that its two domains have swung together

relative to those in the 50% identical yeast PGK (Fig. 17-

15b). This forms the catalytic site and permits the sub-

strates to react in a water-free environment, much as oc-

curs with hexokinase (Section 17-2Aa).

Figure 17-16 indicates a reaction mechanism for PGK

that is consistent with its observed sequential kinetics. The

terminal phosphoryl oxygen of ADP nucleophilically at-

tacks the C1 phosphorus atom of 1,3-BPG to form the re-

action product.

The energetics of the overall GAPDH–PGK reaction

pair are

¢G°¿ ⫽⫺12.1 kJ ⴢ mol

⫺1

GAP ⫹ P

⫹ NAD

⫹

⫹ ADP

3PG ⫹ NADH ⫹ ATP

¢G°¿ ⫽⫺18.8 kJ ⴢ mol

⫺1

1,3-BPG ⫹ ADP

3PG ⫹ ATP

¢G°¿ ⫽ ⫹6.7 kJ ⴢ mol

⫺1

GAP ⫹ P

⫹ NAD

⫹

1,3-BPG ⫹ NADH

Section 17-2. The Reactions of Glycolysis 609

Figure 17-15 X-ray structures of phosphoglycerate kinase

(PGK). (a) The yeast enzyme in complex with 3PG and

2⫹

–ATP. (b) The T. maritima enzyme in complex with 3PG

and Mg

2⫹

–AMPPNP. In both structures, the enzyme is

represented by its transparent molecular surface with its

embedded ribbon diagram colored with its N-terminal domain

yellow and its C-terminal domain purple. The Mg

2⫹

–ATP,

2⫹

–AMPPNP, and 3PG are drawn in space-filling form

Figure 17-16 Mechanism of the PGK reaction.

1,3-Bisphosphoglycerate

3-Phosphoglycerate

2ⴙ

–ADP

2⫹

2ⴙ

–ATP

⫹

OOOP

⫺

CHOH

OPO

2⫺

⫺

OOP

⫺

OP Adenosine

CHOH

OPO

2⫺

2⫹

⫺

O OP

⫺

O O

⫺

Adenosine

⫺

(a)

(b)

colored according to atom type (ATP and AMPPNP C green,

3PG C cyan, N blue, O red, P orange, and Mg

2⫹

pale green). Note

the similar overall appearance of PGK and hexokinase (Fig.

17-5), although these proteins unrelated. [Based on X-ray

structures by Herman Watson, University of Bristol, U.K.; and

Günter Auerbach and Robert Huber, Max-Planck-Institut für

Biochemie, Martinsreid, Germany. PDBids 3PGK and 1VPE.]

JWCL281_c17_593-637.qxd 6/3/10 8:36 AM Page 609

Although the GAPDH reaction is endergonic, the

strongly exergonic nature of the transfer of a phosphoryl

group from 1,3-BPG to ADP makes the overall synthesis

of NADH and ATP from GAP, P

,NAD

⫹

, and ADP

favorable.

H. Phosphoglycerate Mutase

In Reaction 8 of glycolysis, 3PG is converted to 2-phospho-

glycerate (2PG) by phosphoglycerate mutase (PGM):

A mutase catalyzes the transfer of a functional group from

one position to another on a molecule. This reaction is a

necessary preparation for the next reaction in glycolysis,

which generates a “high-energy” phosphoryl compound for

use in ATP synthesis.

–

HOH

3-Phosphoglycerate

(3PG)

OPO

–

O O

–

2-Phosphoglycerate

(2PG)

OPO

–

phosphoglycerate

mutase (PGM)

a. Reaction Mechanism of PGM

At first sight, the reaction catalyzed by PGM appears to

be a simple intramolecular phosphoryl transfer.This is not

the case, however. The active enzyme has a phosphoryl

group at its active site, which it transfers to the substrate to

form a bisphospho intermediate. This intermediate then

rephosphorylates the enzyme to form the product and re-

generate the active phosphoenzyme. The following experi-

mental data permitted the elucidation of PGM’s enzymatic

mechanism:

1. Catalytic amounts of 2,3-bisphosphoglycerate (2,3-

BPG; previously known as 2,3-diphosphoglycerate)

are required for enzymatic activity; that is, 2,3-BPG acts as

a reaction primer.

2. Incubation of the enzyme with catalytic amounts of

P-labeled 2,3-BPG yields a

P-labeled enzyme. Zelda

Rose demonstrated that this was a result of the phosphoryl-

ation of a His residue:

3. The enzyme’s X-ray structure shows His at the active

site (Fig. 17-17). In the active enzyme, His 8 is phosphoryl-

ated.

These data are consistent with a mechanism in which

the active enzyme contains a phospho-His residue at the

active site (Fig. 17-18):

Step 1 3PG binds to the phosphoenzyme in which His 8

is phosphorylated.

Step 2 This phosphoryl group is transferred to the sub-

strate, resulting in an intermediate 2,3-BPG ⴢ enzyme

complex.

Steps 3 and 4 The complex decomposes to form the

product 2PG with regeneration of the phosphoenzyme.

The phosphoryl group on 3PG therefore ends up on the C2

of the next 3PG to undergo reaction.

Enzyme

–

Phospho-His residue

HOPO

2⫺

OPO

2⫺

⫺

2,3-Bisphosphoglycerate

(2,3-BPG)

610 Chapter 17. Glycolysis

Figure 17-17 The active site region of yeast phosphoglycerate

mutase (dephospho form). The substrate, 3PG, which is drawn in

ball-and-stick form with C green, O red, and P orange, binds to

an ionic pocket whose side chains are drawn in stick form with C

green, N blue, and O red. His 8 is phosphorylated in the active

enzyme. [Based on an X-ray structure by Jennifer Littlechild,

University of Exeter, U.K. PDBid 1QHF.]

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