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

(a)

Enzyme main chain

CH2OPO23–

H2N

Lys

CH2OPO23–

CH2OPO23–

C

O

C

H

+

C

H+

H

O

N

N

C

Cys

H O

C

H

H O

C

H

HOCH

Cys

S'

Cys

+ H C OH

–

H

C

O H

H2O

S'

H

C

O

H

HS

CH2OPO23–

H

H

C

OH

H

C

B

OH

G-3-P

CH2OPO2–3

CH2OPO2–3

H2O

(b)

CH2OPO23–

CH2OPO23–

O ... Zn2+

E

O– ... Zn2+

E

FBP

C

C

C–

G-3-P

C

H

HO

H

HO

FIGURE 19.13 ● (a) A mechanism for the fructose-1,6-bisphosphate aldolase reaction. The Schiff base formed between the substrate carbonyl and an active-site lysine acts as an electron sink, increasing the acidity of the -hydroxyl group and facilitating cleavage as shown. (B) In class II aldolases, an active-site Zn2 stabilizes the enolate intermediate, leading to polarization of the substrate carbonyl group.

energetically unfavorable, the overall five-step reaction sequence has a net G° of 2.2 kJ/mol (Keq 0.43). It is the free energy of hydrolysis from the two priming molecules of ATP that brings the overall equilibrium constant close to 1 under standard-state conditions. The net G under cellular conditions is quite negative ( 53.4 kJ/mol in erythrocytes).

CH2OPO23–

C O

CH2OH

DHAP

					H			O
					Triose
CH2OH					phosphate		C
					isomerase
							HCOH
C			O
CH2OPO32–							CH2OPO32–
DHAP							G-3-P
				∆ G° = +7.56 kJ/mol

O

E H

–

Glu O H C OH

...... +

C O H ..B

CH2OPO23–

DHAP

E

O

FIGURE 19.14 ●

The triose phosphate iso-

C

+

merase reaction.

Glu O

H

O

H

..B

E

H

......

C

C

OH

E

CH2OPO23–

Enediol

intermediate

E

O

H

O

C

C

Glu

_

H

C

OH

O

CH2OPO23–

FIGURE 19.15 ●

A reaction mechanism for

Glyceraldehyde-3-P

triose phosphate isomerase.

621

622 Chapter 19 ● Glycolysis

A D E E P E R L O O K

The Chemical Evidence for the Schiff Base Intermediate in Class I Aldolases

Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imine intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate.

These observations are explained by the mechanism shown in the figure. NaBH4 inactivates Class I aldolases by transfer of a hydride ion (H: ) to the imine carbon atom of the enzyme–sub- strate adduct. The resulting secondary amine is stable to hydrolysis, and the active-site lysine is thus permanently modified and inactivated. NaBH4 inactivates Class I aldolases in the presence of either dihydroxyacetone-P or fructose-1,6-bisP, but inhibition doesn’t occur in the presence of glyceraldehyde-3-P.

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from 14C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with 14C. Acid hydrolysis of the inactivated enzyme liberates a novel 14C-labeled amino acid, N 6-dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the 14C-labeled dihydroxy- acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of 14C labeling in a case such as this facilitates the separation and identification of the telltale amino acid.

CH2

OH

+ H2N

C

O

Lys

PO32–

CH2

O

Formation of Schiff base intermediate

CH2

OH

+

C

N

H

Lys

–

H

H

B

H

PO32–

CH2

O

HBorohydride reduction

of Schiff base intermediate

CH2 OH

H C N Lys

H

CH2 O PO32–

Stable (trapped) E–S derivative

Degradation of enzyme (acid hydrolysis)

		CH2							OH
											Lys
H		C					N
							H
		CH2							OH

N6- dihydroxypropyl-L-lysine

19.4 ● The Second Phase of Glycolysis

The second half of the glycolytic pathway involves the reactions that convert the metabolic energy in the glucose molecule into ATP. Altogether, four new ATP molecules are produced. If two are considered to offset the two ATPs consumed in phase 1, a net yield of 2 ATPs per glucose is realized. Phase II starts with the oxidation of glyceraldehyde-3-phosphate, a reaction with a large

19.4 ● The Second Phase of Glycolysis

623

H

O

C

D-Glyceraldehyde-3-phosphate

3,4

H

C

OH

(G3P)

2,5

OPO2–

Glucose

1,6

CH

2

3

ATP

first

P

ADP

priming

D-Glyceraldehyde-3-

reaction

NAD+

(G6P)

phosphate dehydrogenase

Glucose-6-phosphate

NADH +

H+

(F6P)

ATP

Fructose-6-phosphate

second

ADP

priming

O

2–

reaction

Fructose-1,6-bisphosphate

In the second phase

C

OPO3

(FBP)

Dihydroxyacetone phosphate

of glycolysis,

3,4

1,3-Bisphosphoglycerate

(DHAP)

glyceraldehyde-3-

H

C

OH

(BPG)

(G3P)

(G3P)

phosphate is converted

2,5

Glyceraldehyde-3-phosphate

Glyceraldehyde-3-phosphate

P

P

to pyruvate.

OPO2–

CH

NAD+

NAD+

1,6

NADH

NADH

2

3

1,3-bisphosphoglycerate

1,3-bisphosphoglycerate

These reactions yield

ADP

first

first

(BPG)

(BPG)

Mg2+

Phosphoglycerate kinase

ADP

ATP-forming

ATP-forming

ADP

4 molecules of ATP,

ATP

reaction

reaction

ATP

ATP

3-phosphoglycerate (3PG)

3-phosphoglycerate (3PG)

2 for each molecule

of pyruvate produced.

2-phosphoglycerate (2PG)

2-phosphoglycerate (2PG)

COO–

H2O

H2O

Phosphoenolpyruvate (PEP)

Phosphoenolpyruvate (PEP)

3,4

ADP

second

second

ADP

H

C

OH

3-Phosphoglycerate

ATP

ATP-forming ATP-forming

ATP

reaction

reaction

2,5

(3PG)

OPO2–

2 Pyruvate

1,6

CH

2

3

Mg2+

Phosphoglycerate mutase

COO–

3,4

OPO32–

H

C

2-Phosphoglycerate

2,5

(2PG)

CH2OH

1,6

K+, Mg2+

Enolase

H2O

COO–

OPO32–

Phosphoenolpyruvate

C

(PEP)

CH2

ADP

K+, Mg2+

ATP

Pyruvate kinase

COO–

C

O Pyruvate

CH3

FIGURE 19.16 ● The second phase of glycolysis. Carbon atoms are numbered to show their original positions in glucose.

enough energy “kick” to produce a high-energy phosphate, namely, 1,3-bis- phosphoglycerate (Figure 19.16). Phosphoryl transfer from 1,3-BPG to ADP to make ATP is highly favorable. The product, 3-phosphoglycerate, is converted via several steps to phosphoenolpyruvate (PEP), another high-energy phosphate. PEP readily transfers its phosphoryl group to ADP in the pyruvate kinase reaction to make another ATP.

624 Chapter 19 ● Glycolysis

Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase

In the first glycolytic reaction to involve oxidation–reduction, glyceraldehyde- 3-phosphate is oxidized to 1,3-bisphosphoglycerate by glyceraldehyde-3-phos- phate dehydrogenase. Although the oxidation of an aldehyde to a carboxylic acid is a highly exergonic reaction, the overall reaction (Figure 19.17) involves both formation of a carboxylic-phosphoric anhydride and the reduction of NAD to NADH and is therefore slightly endergonic at standard state, with aG° of 6.30 kJ/mol. The free energy that might otherwise be released as heat in this reaction is directed into the formation of a high-energy phosphate compound, 1,3-bisphosphoglycerate, and the reduction of NAD . The reaction mechanism involves nucleophilic attack by a cysteine OSH group on the carbonyl carbon of glyceraldehyde-3-phosphate to form a hemithioacetal (Figure 19.18). The hemithioacetal intermediate decomposes by hydride (H ) transfer to NAD to form a high-energy thioester. Nucleophilic attack by phosphate displaces the product, 1,3-bisphosphoglycerate, from the enzyme. The enzyme can be inactivated by reaction with iodoacetate, which reacts with and blocks the essential cysteine sulfhydryl.

The glyceraldehyde-3-phosphate dehydrogenase reaction is the site of action of arsenate (AsO43 ), an anion analogous to phosphate. Arsenate is an effective substrate in this reaction, forming 1-arseno-3-phosphoglycerate (Figure 19.19), but acyl arsenates are quite unstable and are rapidly hydrolyzed. 1-Arseno-3-phosphoglycerate breaks down to yield 3-phosphoglycerate, the product of the seventh reaction of glycolysis. The result is that glycolysis continues in the presence of arsenate, but the molecule of ATP formed in reaction 7 (phosphoglycerate kinase) is not made because this step has been bypassed. The lability of 1-arseno-3-phosphoglycerate effectively uncouples the oxidation and phosphorylation events, which are normally tightly coupled in the glycer- aldehyde-3-phosphate dehydrogenase reaction.

Reaction 7: Phosphoglycerate Kinase

The glycolytic pathway breaks even in terms of ATPs consumed and produced with this reaction. The enzyme phosphoglycerate kinase transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form an ATP (Figure 19.20). Because each glucose molecule sends two molecules of glyceraldehyde-3-phos- phate into the second phase of glycolysis and because two ATPs were consumed per glucose in the first-phase reactions, the phosphoglycerate kinase reaction “pays off” the ATP debt created by the priming reactions. As might be expected for a phosphoryl transfer enzyme, Mg2 ion is required for activity, and the true nucleotide substrate for the reaction is MgADP . It is appropriate to view the sixth and seventh reactions of glycolysis as a coupled pair, with 1,3-bis-

H

O

O

C

CO

PO23–

+ NADH + H+

HCOH

+ NAD+ + HPO24–

HCOH

CH2O

PO23–

CH2O

PO23–

Glyceraldehyde-

1,3-Bisphosphoglycerate

3-phosphate

1,3-BPG

G3P

∆ G ' = +6.3 kJ/mol

FIGURE 19.17 ● The glyceraldehyde-3-phosphate dehydrogenase reaction.

FIGURE 19.18

H

C O

E		SH	HCOH

CH2OPO23–

O OPO23–

C

HCOH

CH2OPO23–

1,3-Bisphosphoglycerate

		H+
					OPO23–
...
								O–
E		S			C
				HCOH
						CH2O			PO23–

R

N +

H2N

O H

E S C O H

HCOH

CH2OPO23–

R

N

NH2

HH O

+O

–O P OH

O

O– E S C

HCOH

CH2O PO23–

19.4 ● The Second Phase of Glycolysis

625

● A mechanism for the glycer- aldehyde-3-phosphate dehydrogenase reaction. Reaction of an enzyme sulfhydryl with the carbonyl carbon of glyceraldehyde-3-P forms a thiohemiacetal, which loses a hydride to NAD to become a thioester. Phosphorolysis of this thioester releases 1,3-bisphosphoglycerate.

phosphoglycerate as an intermediate. The phosphoglycerate kinase reaction is sufficiently exergonic at standard state to pull the G-3-P dehydrogenase reaction along. (In fact, the aldolase and triose phosphate isomerase are also pulled forward by phosphoglycerate kinase.) The net result of these coupled reactions is

Glyceraldehyde-3-phosphate ADP Pi NAD 88n

3-phosphoglycerate ATP NADH H

G° 12.6 kJ/mol

(19.9)

O

COO –

C

O

PO23–

+ ADP

Mg2+

+

ATP

HCOH

HCOH

Phosphoglycerate

CH2O

PO23–

kinase

CH2O

PO23–

1,3-Bisphosphoglycerate

3-Phosphoglycerate

(1,3-BPG)

(3-PG)

∆ G ' = –18.9 kJ/mol

FIGURE 19.20 ● The phosphoglycerate kinase reaction.

							O
O				O						O–
							As
		C
							O–
H		C			OH

CH2OPO23–

1-Arseno-3-phosphoglycerate

FIGURE 19.19

626 Chapter 19 ● Glycolysis

O

C OPO23–

H C OH

H C OPO23–

H

1,3-Bisphosphoglycerate

(1,3-BPG)

O

O

H+

C

O–

H2O

P + H+

C

O–

Bisphosphoglycerate

H

C

OPO23–

H

C

OH

2,3-Bisphosphoglycerate H

H

C

OPO23–

C

OPO23–

mutase

phosphatase

H

H

2,3-Bisphosphoglycerate

3-Phosphoglycerate

(2,3-BPG)

FIGURE 19.21 ● Formation and decomposition of 2,3-bisphosphoglycerate.

Another reflection of the coupling between these reactions lies in their values of G under cellular conditions (Table 19.1). In spite of its strongly negative G° , the phosphoglycerate kinase reaction operates at equilibrium in the erythrocyte ( G 0.1 kJ/mol). In essence, the free energy available in the phosphoglycerate kinase reaction is used to bring the three previous reactions closer to equilibrium. Viewed in this context, it is clear that ADP has been phosphorylated to form ATP at the expense of a substrate, namely, glyceraldehyde- 3-phosphate. This is an example of substrate-level phosphorylation, a concept that will be encountered again. (The other kind of phosphorylation, oxidative phosphorylation, is driven energetically by the transport of electrons from appropriate coenzymes and substrates to oxygen. Oxidative phosphorylation will be covered in detail in Chapter 21). Even though the coupled reactions exhibit a very favorable G° , there are conditions (i.e., high ATP and 3-phosphoglyc- erate levels) under which Equation 19.9 can be reversed, so that 3-phospho- glycerate is phosphorylated from ATP.

An important regulatory molecule, 2,3-bisphosphoglycerate, is synthesized and metabolized by a pair of reactions that make a detour around the phosphoglycerate kinase reaction. 2,3-BPG, which stabilizes the deoxy form of hemoglobin and is primarily responsible for the cooperative nature of oxygen binding by hemoglobin (see Chapter 15), is formed from 1,3-bisphosphoglycerate by bisphosphoglycerate mutase (Figure 19.21). Interestingly, 3-phosphoglycer- ate is required for this reaction, which involves phosphoryl transfer from the C-1 position of 1,3-bisphosphoglycerate to the C-2 position of 3-phosphoglyc- erate (Figure 19.22). Hydrolysis of 2,3-BPG is carried out by 2,3-bisphosphoglyc- erate phosphatase. Although other cells contain only a trace of 2,3-BPG, erythrocytes typically contain 4 to 5 mM 2,3-BPG.

Reaction 8: Phosphoglycerate Mutase

The remaining steps in the glycolytic pathway prepare for synthesis of the second ATP equivalent. This begins with the phosphoglycerate mutase reaction (Figure 19.23), in which the phosphoryl group of 3-phosphoglycerate is moved

1	P	1		1		1
2		+ 2		2		+ 2	P
3	P	3	P	3	P	3	P

FIGURE 19.22 ● The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglyc- erate. The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-PG.

FIGURE 19.24

FIGURE 19.23 mutase reaction.

from C-3 to C-2. (The term mutase is applied to enzymes that catalyze migration of a functional group within a substrate molecule.) The free energy change for this reaction is very small under cellular conditions ( G 0.83 kJ/mol in erythrocytes). Phosphoglycerate mutase enzymes isolated from different sources exhibit different reaction mechanisms. As shown in Figure 19.24, the

Enzyme

αε

+

NH3

+

NH3

	Phosphohistidine
		O
	–O	P	+
B			N NH
	H	O
	O
–O		H	H
O	H
		O	O
			P	N NH
		O–	O–

3-Phosphoglycerate (3-PG)

	O	O–
	P		N	NH
	O	O–
–O	H	H
O	H
	O		O
	+	P	N	NH
	B:H
	– O		O–

2,3-Bisphosphoglycerate intermediate

		O	O–
			P	N	NH
+		O	O–	Phosphohistidine
NH3	–O		H
			H		O
	O	H	OH	–O	P N NH
					O –

2-Phosphoglycerate (2-PG)

19.4 ● The Second Phase of Glycolysis

627

COO–						COO–
HCOH						HCO		PO23–
CH2O		PO23–				CH2OH
3-Phosphoglycerate					2-Phosphoglycerate
	(3-PG)						(2-PG)

∆ G ' = +4.4 kJ/mol

● The phosphoglycerate

● A mechanism for the phosphoglycerate mutase reaction in rabbit muscle and in yeast. Zelda Rose of the Institute for Cancer Research in Philadelphia showed that the enzyme requires a small amount of 2,3-BPG to phosphorylate the histidine residue before the mechanism can proceed. Prior to her work, the role of the phosphohistidine in this mechanism was not understood.

FIGURE 19.25

628 Chapter 19 ● Glycolysis

HC

H2

Enzyme

COO–	COO–	CO2
	HC	HC O P
H2	H2	H2COH

● The phosphoglycerate mutase of wheat germ catalyzes an intramolecular phosphoryl transfer.

enzymes isolated from yeast and from rabbit muscle form phosphoenzyme intermediates, use 2,3-bisphosphoglycerate as a cofactor, and undergo intermolecular phosphoryl group transfers (in which the phosphate of the product 2-phos- phoglycerate is not that from the 3-phosphoglycerate substrate). The prevalent form of phosphoglycerate mutase is a phosphoenzyme, with a phosphoryl group covalently bound to a histidine residue at the active site. This phosphoryl group is transferred to the C-2 position of the substrate to form a transient, enzymebound 2,3-bisphosphoglycerate, which then decomposes by a second phosphoryl transfer from the C-3 position of the intermediate to the histidine residue on the enzyme. About once in every 100 enzyme turnovers, the intermediate, 2,3-bisphosphoglycerate, dissociates from the active site, leaving an inactive, unphosphorylated enzyme. The unphosphorylated enzyme can be reactivated by binding 2,3-BPG. For this reason, maximal activity of phosphoglycerate mutase requires the presence of small amounts of 2,3-BPG.

A different mechanism operates in the wheat germ enzyme. 2,3- Bisphosphoglycerate is not a cofactor. Instead, the enzyme carries out intra- molecular phosphoryl group transfer (Figure 19.25). The C-3 phosphate is transferred to an active-site residue and then to the C-2 position of the original substrate molecule to form the product, 2-phosphoglycerate.

Reaction 9: Enolase

Recall that, prior to synthesizing ATP in the phosphoglycerate kinase reaction, it was necessary to first make a substrate having a high-energy phosphate. Reaction 9 of glycolysis similarly makes a high-energy phosphate in preparation for ATP synthesis. Enolase catalyzes the formation of phosphoenolpyruvate from 2-phosphoglycerate (Figure 19.26). The reaction in essence involves a dehydration—the removal of a water molecule—to form the enol structure of PEP. The G° for this reaction is relatively small at 1.8 kJ/mol (Keq 0.5); and, under cellular conditions, G is very close to zero. In light of this condition, it may be difficult at first to understand how the enolase reaction transforms a substrate with a relatively low free energy of hydrolysis into a product (PEP) with a very high free energy of hydrolysis. This puzzle is clarified by real-

COO–

COO–

Mg2+

+ H2O

H

C

O

PO23–

C

O

PO23–

CH2

OH

CH2

2-Phosphoglycerate	Phosphoenolpyruvate
(2-PG)	(PEP)
	∆ G ' = +1.8 kJ/mol

FIGURE 19.26 ● The enolase reaction.

19.4 ● The Second Phase of Glycolysis

629

COO–

Mg2+

COO–

O

PO23–

+ H+ + ADP3–

O + ATP 4–

C

C

K+

C H2

C H3

PEP

Pyruvate

∆ G ' = –31.7 kJ/mol

FIGURE 19.27 ● The pyruvate kinase reaction.

izing that 2-phosphoglycerate and PEP contain about the same amount of potential metabolic energy, with respect to decomposition to Pi, CO2, and H2O. What the enolase reaction does is rearrange the substrate into a form from which more of this potential energy can be released upon hydrolysis. The enzyme is strongly inhibited by fluoride ion in the presence of phosphate. Inhibition arises from the formation of fluorophosphate (FPO32 ), which forms a complex with Mg2 at the active site of the enzyme.

Reaction 10: Pyruvate Kinase

The second ATP-synthesizing reaction of glycolysis is catalyzed by pyruvate kinase, which brings the pathway at last to its pyruvate branch point. Pyruvate kinase mediates the transfer of a phosphoryl group from phosphoenolpyruvate to ADP to make ATP and pyruvate (Figure 19.27). The reaction requires Mg2 ion and is stimulated by K and certain other monovalent cations.

		G° (kJ/mol)
Phosphoenolpyruvate3 H2O 88n pyruvate HPO42		62.2
ADP3 HPO42 H 88n ATP4 H2O		30.5
Phosphoenolpyruvate3 ADP3 H 88n pyruvate ATP4		31.7

The corresponding Keq at 25°C is 3.63 105, and it is clear that the pyruvate kinase reaction equilibrium lies very far to the right. Concentration effects reduce the magnitude of the free energy change somewhat in the cellular environment, but the G in erythrocytes is still quite favorable at 23.0 kJ/mol. The high free energy change for the conversion of PEP to pyruvate is due largely to the highly favorable and spontaneous conversion of the enol tautomer of pyruvate to the more stable keto form (Figure 19.28) following the phosphoryl group transfer step.

–OOC

O

PO32–

–OOC

O

H

–OOC

O

C

C

C

C

C ......H+

H

C

H

ADP ATP

H

H

H

H

H

PEP

Enol

Keto

tautomer

tautomer

Pyruvate

FIGURE 19.28 ● The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer followed by an enol-keto tautomerization. The tautomerization is spontaneous ( G° 35–40 kJ/mol) and accounts for much of the free energy change for PEP hydrolysis.

630 Chapter 19 ● Glycolysis

FIGURE 19.29 ● A mechanism for the pyruvate kinase reaction, based on NMR and EPR studies by Albert Mildvan and colleagues.

Phosphoryl transfer from phosphoenolpyruvate (PEP) to ADP occurs in four steps: (a) a water on the Mg2 ion coordinated to ADP is replaced by the phosphoryl group of PEP; (b) Mg2 dissociates from the -P of ADP; (c) the phosphoryl group is transferred; and (d) the enolate of pyruvate is protonated. (Adapted from

Mildvan, A., 1979. Advances in Enzymology 49:103–126.)

The large negative G of this reaction makes pyruvate kinase a suitable target site for regulation of glycolysis. For each glucose molecule in the glycolysis pathway, two ATPs are made at the pyruvate kinase stage (because two triose molecules were produced per glucose in the aldolase reaction). Because the pathway broke even in terms of ATP at the phosphoglycerate kinase reaction (two ATPs consumed and two ATPs produced), the two ATPs produced by pyruvate kinase represent the “payoff” of glycolysis—a net yield of two ATP molecules.

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the -amino acid counterpart of the -keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Hormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phosphorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher Km for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the gluconeogenesis pathway (to be described in Chapter 23), instead of going on through glycolysis and the citric acid cycle (or fermentation routes). A suggested active-site geometry for pyruvate kinase, based on NMR and EPR studies by Albert Mildvan and colleagues, is presented in Figure 19.29. The carbonyl oxygen of pyruvate and the -phosphorus of ATP lie within 0.3 nm of each other at the active site, consistent with direct transfer of the phosphoryl group without formation of a phosphoenzyme intermediate.

	O		M+
		O
		C
					Mg2+
H		C	O			O	O
	C			H O	Mg2+		Adenine
		O P					P
	H		O	H			Ribose
			O
							O
	H
	B			O	P	O

O

	O		M+
		O
		C
H		C			Mg2+
						–
	C						O
H			O		Mg2+	O
		O		O			Adenine
							P
	H
			P		H2O		Ribose
	B	O					O
				O	P	O

O