Biochemistry, 4th Edition P59 docx

Reaction 5: Triose Phosphate Isomerase Completes the First Phase of Glycolysis Of the two products of the aldolase reaction, only glyceraldehyde-3-phosphate goes directly into the second

Adenylate kinase rapidly interconverts ADP, ATP, and AMP to maintain this

equi-librium ADP levels in cells are typically 10% of ATP levels, and AMP levels are

often less than 1% of the ATP concentration Under such conditions, a small net

change in ATP concentration due to ATP hydrolysis results in a much larger

rela-tive increase in the AMP levels because of adenylate kinase activity

Clearly, the activity of phosphofructokinase depends on both ATP and AMP levels

and is a function of the cellular energy status Phosphofructokinase activity is

in-creased when the energy status falls and is dein-creased when the energy status is high

The rate of glycolysis activity thus decreases when ATP is plentiful and increases when

more ATP is needed

Glycolysis and the citric acid cycle (to be discussed in Chapter 19) are coupled via

phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an

allosteric inhibitor of phosphofructokinase When the citric acid cycle reaches

satu-ration, glycolysis (which “feeds” the citric acid cycle under aerobic conditions) slows

down The citric acid cycle directs electrons into the electron-transport chain (for the

purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor

molecules for biosynthetic pathways Inhibition of glycolysis by citrate ensures

that glucose will not be committed to these activities if the citric acid cycle is already

saturated

Phosphofructokinase is also regulated by -D-fructose-2,6-bisphosphate, a

po-tent allosteric activator that increases the affinity of phosphofructokinase for the

substrate fructose-6-phosphate (Figure 18.10) Stimulation of

phosphofructo-kinase is also achieved by decreasing the inhibitory effects of ATP (Figure 18.11)

Fructose-2,6-bisphosphate increases the net flow of glucose through glycolysis by

stimulating phosphofructokinase and, as we shall see in Chapter 22, by inhibiting

fructose-1,6-bisphosphatase, the enzyme that catalyzes this reaction in the opposite

direction

Reaction 4: Cleavage by Fructose Bisphosphate Aldolase Creates

Two 3-Carbon Intermediates

Fructose bisphosphate aldolase cleaves fructose-1,6-bisphosphate between the

C-3 and C-4 carbons to yield two triose phosphates The products are

dihydroxy-acetone phosphate (DHAP) and glyceraldehyde-3-phosphate The reaction has an

equilibrium constant of approximately 104 M, and a corresponding G° of

23.9 kJ/mol These values might imply that the reaction does not proceed

ef-fectively from left to right as written However, the reaction makes two molecules

(glyceraldehyde-3-P and dihydroxyacetone-P) from one molecule

(fructose-1,6-bisphosphate), and the equilibrium is thus greatly influenced by concentration

A DEEPER LOOK

Phosphoglucoisomerase—A Moonlighting Protein

When someone has a day job but also works at night (that is,

un-der the moon) at a second job, they are said to be

“moonlight-ing.” Similarly, a number of proteins have been found to have

two or more different functions, and Constance Jeffery at

Bran-deis University has dubbed these “moonlighting proteins.”

Phos-phoglucoisomerase catalyzes the second step of glycolysis but

also moonlights as a nerve growth factor outside animal cells In

fact, outside the cell, this protein is known as neuroleukin (NL),

autocrine motility factor (AMF), and differentiation and

matu-ration mediator (DMM) Neuroleukin is secreted by (immune

system) T cells and promotes the survival of certain spinal

neu-rons and sensory nerves AMF is secreted by tumor cells and

stimulates cancer cell migration DMM causes certain leukemia cells to differentiate

How phosphoglucoisomerase is secreted by the cell for its moonlighting functions is unknown, but there is evidence that the organism itself may be harmed by this secretion Diane Mathis and Christophe Benoist at the University of Strasbourg have shown that, in mice with disorders similar to rheumatoid arthritis, the im-mune system recognizes extracellular phosphoglucoisomerase as

an antigen—that is, a protein that is “nonself.” That a protein can

be vital to metabolism inside the cell and also function as a growth factor and occasionally act as an antigen outside the cell is indeed remarkable

CH2OH

2 –O3POCH2 O OPO3–

H

Fructose-2,6-bisphosphate

100

20

[Fructose-6-phosphate] (

40 60 80

0

0

0.1 1.0

FIGURE 18.10 Fructose-2,6-bisphosphate activates phos-phofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic de-pendence of enzyme activity on substrate concentra-tion.

544 Chapter 18 Glycolysis

The value of G in erythrocytes is actually 0.23 kJ/mol (see Table 18.1) At

phys-iological concentrations, the reaction is essentially at equilibrium

Two classes of aldolase enzymes are found in nature Animal tissues produce a Class I aldolase, characterized by the formation of a covalent Schiff base intermediate between an active-site lysine and the carbonyl group of the substrate Class I aldolases

do not require a divalent metal ion Class II aldolases are produced mainly in bacte-ria and fungi and do not form a covalent E-S intermediate, but they contain an active-site metal (normally zinc, Zn2) Cyanobacteria and some other simple organisms possess both classes of aldolase

The aldolase reaction is merely the reverse of the aldol condensation well known

to organic chemists The latter reaction involves an attack by a nucleophilic enolate anion of an aldehyde or ketone on the carbonyl carbon of an aldehyde The oppo-site reaction, aldol cleavage, begins with removal of a proton from the -hydroxyl

group, which is followed by the elimination of the enolate anion A mechanism for the aldol cleavage reaction of fructose-1,6-bisphosphate in the Class I–type aldolases

is shown in Figure 18.12a In Class II aldolases, an active-site metal such as Zn2 be-haves as an electrophile, polarizing the carbonyl group of the substrate and stabiliz-ing the enolate intermediate (Figure 18.12b)

Reaction 5: Triose Phosphate Isomerase Completes the First Phase of Glycolysis

Of the two products of the aldolase reaction, only glyceraldehyde-3-phosphate goes directly into the second phase of glycolysis The other triose phosphate, dihydroxyacetone phosphate, must be converted to glyceraldehyde-3-phosphate by

the enzyme triose phosphate isomerase This reaction thus permits both products of

the aldolase reaction to continue in the glycolytic pathway and in essence makes the C-1, C-2, and C-3 carbons of the starting glucose molecule equivalent to the C-6, C-5, and C-4 carbons, respectively The reaction mechanism involves an enediol in-termediate that can donate either of its hydroxyl protons to a basic residue on the enzyme and thereby become either dihydroxyacetone phosphate or glyceraldehyde-3-phosphate (Figure 18.13) Triose phosphate isomerase is one of the enzymes that have evolved to a state of “catalytic perfection,” with a turnover number near the dif-fusion limit (see Table 13.5)

The triose phosphate isomerase reaction completes the first phase of glycolysis, each glucose that passes through being converted to two molecules of glyceralde-hyde-3-phosphate Although the last two steps of the pathway are energetically un-favorable, 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

[ATP] (

0.1

0

1.0

0

FIGURE 18.11 Fructose-2,6-bisphosphate decreases the

inhibition of phosphofructokinase due to ATP.

C

C

C

Aldol cleavage

PO3–

CH2O

CH2OH

C O H

PO3–

CH2O

PO3–

CH2O

PO3–

CH2O

D -Fructose-1,6-bisphosphate

(FBP)

Fructose bisphosphate aldolase

Dihydroxyacetone phosphate (DHAP)

D -Glyceraldehyde 3-phosphate (G-3-P)

ΔG°' = 23.9 kJ/mol

O

H

R

C

O

R  H H R H

R  = H (aldehyde)

R = alkyl, etc (ketone)

Aldol condensation

R 

H

C HCOH

CH2OPO3–

G-3-P DHAP

CH2OPO3–

CH2OH

ΔG° = +7.56 kJ/mol

Triose

phosphate

isomerase

CH2OPO32–

O

H2N

C O

O

– O H

C

HOCH

H

N

C O

C O

G-3-P

DHAP

Lys 229

–

H+

+

CH2OPO32–

H2O

Schiff base formation

Schiff base hydrolysis

CH2OPO32–

CH2OPO32–

CH2OPO32–

CH2OPO32–

CH2OPO32–

CH2OH

H 2 O

Asp33

C O

O

– O Asp33

Asp 33

Lys229

H

C

HO

H2N Lys 229

Asp 33 – O

Enzyme main chain

(a)

CH2OPO3–

C O

C– H HO

Zn 2 +

CH2OPO3–

C O– C

H HO

Zn 2 +

G-3-P

(b)

ACTIVE FIGURE 18.12 (a) A

mecha-nism for the fructose-1,6-bisphosphate aldolase reac-tion 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 The catalytic residues in the rabbit muscle enzyme are Lys 229 and Asp 33 (b) In

Class II aldolases, an active-site Zn2stabilizes the eno-late intermediate, leading to polarization of the

sub-strate carbonyl group Test yourself on the concepts

in this figure at www.cengage.com/login.

H

CH2OPO3–

O O

H B +

Glu 165

O

O

H

CH2OPO3–

C

C H

H

CH2OPO3–

C

O_

Glu

C

– E

E

E

E

Glyceraldehyde-3-P

Enediol intermediate DHAP

ACTIVE FIGURE 18.13 A reaction mecha-nism for triose phosphate isomerase In the enzyme from yeast, the catalytic residue is Glu 165 Test yourself on the concepts in this figure at www.cengage.com/login.

Triose phosphate isomerase with substrate analog

2-phosphoglycerate shown in cyan (pdb id  1YPI).

Glu 165

546 Chapter 18 Glycolysis

conditions The net G under cellular conditions is quite negative (53.4 kJ/mol

in erythrocytes)

of 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 mol-ecules are produced If two are considered to offset the two ATPs consumed in phase 1, a net yield of two ATPs per glucose is realized Phase 2 starts with the oxi-dation of glyceraldehyde-3-phosphate, a reaction with a large enough energy “kick”

to produce a high-energy phosphate, namely, 1,3-bisphosphoglycerate (see Figure 18.1) Phosphoryl transfer from 1,3-BPG to ADP to make ATP is highly favorable The product, 3-phosphoglycerate, is converted via several steps to phosphoenol-pyruvate (PEP), another high-energy phosphate PEP readily transfers its phos-phoryl group to ADP in the pyruvate kinase reaction to make another ATP

Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase Creates

a High-Energy Intermediate

In the first glycolytic reaction to involve oxidation–reduction,

glyceraldehy3-phosphate is oxidized to 1,3-bisphosphoglycerate by glyceraldehy3-glyceraldehy3-phosphate

de-hydrogenase.Although the oxidation of an aldehyde to a carboxylic acid is a highly exergonic reaction, the overall reaction involves both formation of a carboxylic– phosphoric anhydride and the reduction of NADto NADH and is therefore slightly endergonic at standard state, with a G° 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 hemithioac-etal (Figure 18.14) The hemithioachemithioac-etal intermediate decomposes by hydride (H⬊) transfer to NADto 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 cys-teine sulfhydryl

O

C HCOH

CH2OPO3–

CH2OPO3–

+

COPO3–

H +

+

NADH NAD +

Glyceraldehyde-3-phosphate (G-3-P)

1,3-Bisphosphoglycerate (1,3-BPG)

ΔG⬚' = +6.3 kJ/mol

The glyceraldehyde-3-phosphate dehydrogenase reaction is the site of action of

arsenate (AsO4 ), an anion analogous to phosphate Arsenate is an effective

sub-strate in this reaction, forming 1-arseno-3- phosphoglycerate, 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 un-couples the oxidation and phosphorylation events, which are normally tightly cou-pled in the glyceraldehyde-3-phosphate dehydrogenase reaction

CH2OPO3–

O

C

O

O–

C

1-Arseno-3-phosphoglycerate

Reaction 7: Phosphoglycerate Kinase Is the Break-Even Reaction

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 Because each

glu-cose molecule sends two molecules of glyceraldehyde-3-phosphate into the

sec-ond 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, Mg2ion is required for activity and the true nucleotide

sub-strate for the reaction is MgADP It is appropriate to view the sixth and seventh

reactions of glycolysis as a coupled pair, with 1,3-bisphosphoglycerate as an

in-termediate The phosphoglycerate kinase reaction is sufficiently exergonic at

standard state to pull the G-3-P dehydrogenase reaction along (In fact, the

H C HCOH

O SH

HCOH

CH2OPO3–

H O

H2N

N

R +

N R

NH2

+

C HCOH

CH2OPO3–

S

O C

HCOH

CH2OPO3–

OPO3–

H+

C HCOH

CH2OPO3–

CH2OPO3–

OPO3– O

E E

1,3-Bisphosphoglycerate

OH O–

H+

P

O –O

H

ACTIVE FIGURE 18.14 A mechanism for the glyceraldehyde-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 In the enzyme from rabbit muscle, the catalytic residue is Cys 149 Test yourself on the concepts in this figure at www.cengage.com/login.

C

O

OPO3–

HCOH

CH2OPO3–

+

CH2OPO3–

–

ATP

ADP

1,3-Bisphosphoglycerate

(1,3-BPG)

3-Phosphoglycerate (3-PG)

ΔG⬚' = –18.9 kJ/mol

Phosphoglycerate kinase

(a)

(b)

The open (a) and closed (b) forms of phosphoglycerate

kinase ATP (cyan), 3-phosphoglycerate (purple), and

Mg 2H (gold) (a: pdb id  3PGK; b: pdb id  1VPE).

548 Chapter 18 Glycolysis

aldolase and triose phosphate isomerase are also pulled forward by phospho-glycerate kinase.) The net result of these coupled reactions is

Glyceraldehyde-3-phosphate ADP  Pi NAD⎯

3-phosphoglycerate ATP  NADH  H

G°  12.6 kJ/mol (18.8) Another reflection of the coupling between these reactions lies in their values

ofG under cellular conditions (Table 18.1) Despite 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 equilib-rium 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

en-countered again (The other kind of phosphorylation, oxidative phosphorylation,

is driven energetically by the transport of electrons from appropriate coen-zymes and substrates to oxygen Oxidative phosphorylation will be covered in detail in Chapter 20.) Even though the coupled reactions exhibit a very favorable

G°, there are conditions (that is, high ATP and 3-phosphoglycerate levels)

under which the phosphoglycerate kinase reaction can be reversed so that 3-phosphoglycerate 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 pri-marily responsible for the cooperative nature of oxygen binding by hemoglobin (see

Chapter 15), is formed from 1,3-bisphosphoglycerate by bisphosphoglycerate mutase

(Figure 18.15) Interestingly, 3-phosphoglycerate 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-phosphoglycerate (Figure 18.16) Hydrolysis of 2,3-BPG is carried

out by 2,3-bisphosphoglycerate 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 Catalyzes a Phosphoryl Transfer

The remaining steps in the glycolytic pathway prepare for synthesis of the second ATP

equivalent This begins with the phosphoglycerate mutase reaction, in which the

phosphoryl group of 3-phosphoglycerate is moved 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 con-ditions (G  0.83 kJ/mol in erythrocytes) Phosphoglycerate mutase enzymes

iso-lated from different sources exhibit different reaction mechanisms As shown in

Fig-ure 18.17, the enzymes isolated from yeast and from rabbit muscle form phosphoenzyme intermediates, use 2,3-bisphosphoglycerate as a cofactor, and undergo inter molecular

phosphoryl group transfers (in which the phosphate of the product 2-phosphoglyc-erate is not that from the 3-phosphoglyc2-phosphoglyc-erate substrate) The prevalent form of

phos-C H

OPO3– O

C H

C

H

OH OPO3–

H +

C H O

C H

C

H

H2O Pi +H +

C H O

C H

C

H

O– OH OPO3–

OPO3– O–

OPO3–

1,3-Bisphosphoglycerate (1,3-BPG)

Bisphosphoglycerate mutase

2,3-Bisphosphoglycerate (2,3-BPG)

3-Phosphoglycerate

2,3-Bisphosphoglycerate phosphatase

FIGURE 18.15 Formation and decomposition of 2,3-bisphosphoglycerate.

3-Phosphoglycerate

(3-PG)

2-Phosphoglycerate (2-PG) Phosphoglycerate mutase

ΔG⬚' = +4.4 kJ/mol

COO–

HCOH

CH2OPO3–

HCOPO3–

CH2OH COO–

phoglycerate 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, enzyme-bound 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

phosphoglyc-erate mutase requires the presence of small amounts of 2,3-BPG

Reaction 9: Dehydration by Enolase Creates PEP

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

Enolasecatalyzes the formation of phosphoenolpyruvate from 2-phosphoglycerate The

reaction involves the removal of a water molecule to form the enol structure of PEP

TheG° 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

dif-ficult 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

+

1

2

3

1 2 3

1 2 3

+

3

1 2

P

P

P

FIGURE 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate The reaction is

actu-ally an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-PG.

P

O N

+

NH

B

Phosphohistidine

H2C

HC

H

P

P

O

O

O

O–

O–

O COO–

H2C

HC OH O COO–

H2C HC

P O

O O

O–

P O O–

O–

COO–

+ BH

P

O N

O–

FIGURE 18.17 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.

His10 His183

Gly 11

Gly184

Glu88

Asn 16 Arg61

Arg9

SO4–

The catalytic histidine (His 183 ) at the active site of

Escherichia coli phosphoglycerate mutase (pdb id  1E58) Note that His 10 is phosphorylated.

COO–

CH2OH

COO–

CH2

+

Mg 2 +

2-Phosphoglycerate

(2-PG)

Phosphoenolpyruvate (PEP)

ΔG⬚' = +1.8 kJ/mol

550 Chapter 18 Glycolysis

energy of hydrolysis This puzzle is clarified by realizing 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 phos-phate Thomas Nowak has shown that fluoride, phosphate, and a divalent cation form a transition-state–like complex in the enzyme active site, with fluoride appar-ently mimicking the hydroxide ion nucleophile in the enolase reaction

Yeast enolase is a dimer of identical subunits However, if the enzyme is crystal-lized in the presence of a mixture of the substrate (2-phosphoglycerate) and the product (phosphoenolpyruvate), the crystallized dimer is asymmetric! One subunit active site contains 2-phosphoglycerate, and the other contains PEP (Figure 18.18), thus providing a “before-and-after” picture of this glycolytic enzyme

Reaction 10: Pyruvate Kinase Yields More ATP

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 me-diates the transfer of a phosphoryl group from phosphoenolpyruvate to ADP to make ATP and pyruvate The reaction requires Mg2ion and is stimulated by K and certain other monovalent cations

The corresponding Keqat 25°C is 3.63 105, and it is clear that the pyruvate ki-nase reaction equilibrium lies very far to the right Concentration effects reduce the magnitude of the free energy change somewhat in the cellular environment, but theG in erythrocytes is still quite favorable at 23.0 kJ/mol The high free energy

His159 His159

Mg

Li

Phosphoglycerate

Mg

PEP

H2O

FIGURE 18.18 The yeast enolase dimer is asymmetric The active site of one subunit (a) contains

2-phosphoglycerate, the enolase substrate Also shown are a Mg 2  ion (blue), a Li  ion (purple), and His 159 ,

which participates in catalysis The other subunit (b) binds phosphoenolpyruvate, the product of the enolase

reaction An active site water molecule (yellow), Mg 2  (blue), and His 159 are also shown (pdb id  2ONE).

COO–

CH2

Mg2+ K+

COO–

CH3

+

ΔG⬚' = –31.7 kJ/mol

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 18.19) following the phosphoryl group transfer step

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

pro-duced 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

phos-phoryl group from ATP to the enzyme The phosphos-phorylated form of pyruvate kinase

is more strongly inhibited by ATP and alanine and has a higher K mfor PEP, so 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 22), instead of going on through glycolysis and the citric acid cycle (or

fer-mentation routes) A suggested active-site geometry for pyruvate kinase, based on

NMR and EPR studies by Albert Mildvan and colleagues, is presented in Figure

18.20 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

phos-phoryl group without formation of a phosphoenzyme intermediate

The structure of the pyruvate kinase tetramer is sensitive to bound ligands The inactive E coli enzyme in the

absence of ligands (left, pdb id  1E0U).The active form of the yeast dimer, with fructose-1,6-bisphosphate (an

allosteric regulator, blue), substrate analog (red), and K(gold) (pdb id  1A3W).

C

C

H H

PEP

C C

H H

H H

H .H+

C C O

ATP

ADP

Enol tautomer

Keto tautomer Pyruvate

FIGURE 18.19 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer followed by an enol–keto tautomer-ization The tautomerization is spontaneous (G° ⬇

35–40 kJ/mol) and accounts for much of the free en-ergy change for PEP hydrolysis.

552 Chapter 18 Glycolysis

Produced in Glycolysis?

In addition to ATP, the products of glycolysis are NADH and pyruvate Their pro-cessing depends upon other cellular pathways NADH must be recycled to NAD, lest NADbecome limiting in glycolysis NADH can be recycled by both aerobic and anaerobic paths, either of which results in further metabolism of pyruvate What a given cell does with the pyruvate produced in glycolysis depends in part on the avail-ability of oxygen Under aerobic conditions, pyruvate can be sent into the citric acid cycle (also known as the TCA cycle; see Chapter 19), where it is oxidized to CO2 with the production of additional NADH (and FADH2) Under aerobic conditions, the NADH produced in glycolysis and the citric acid cycle is reoxidized to NADin the mitochondrial electron-transport chain (see Chapter 20)

Anaerobic Metabolism of Pyruvate Leads to Lactate or Ethanol

Under anaerobic conditions, the pyruvate produced in glycolysis is processed differ-ently In yeast, it is reduced to ethanol; in other microorganisms and in animals, it is

reduced to lactate These processes are examples of fermentation—the production of

ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons In either case, reduction of pyruvate provides a means of re-oxidizing the NADH produced in the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis (Figure 18.21) In yeast, alcoholic fermentation is a two-step

M+

O

O

O O

O P P

O M+

O O

O

B H H

H

H

C C

C

C C

C

Mg2+

H

B

–

O

P

P

O

P

O

O

O

O

O

O

Mg2+

O O

O

O

H

Mg2+

H

H

O

O

Mg2+

O

Adenine Ribose

Adenine Ribose

H2O

FIGURE 18.20 A mechanism for the pyruvate kinase

reaction, based on NMR and EPR studies by Albert

Mildvan and colleagues Phosphoryl transfer from

phos-phoenolpyruvate (PEP) to ADP occurs in four steps:

(1) A water on the Mg2ion coordinated to ADP is

replaced by the phosphoryl group of PEP, (2) Mg2

dis-sociates from the -P of ADP, (3) the phosphoryl group

is transferred, and (4) the enolate of pyruvate is

proto-nated (Adapted from Mildvan, A., 1979 The role of metals in

enzyme-catalyzed substitutions at each of the phosphorus

atoms of ATP Advances in Enzymology 49:103–126.)