Voet D., Voet Ju.G. Biochemistry

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self-limiting: Individuals with fructose intolerance rapidly

develop a strong distaste for anything sweet.

B. Galactose

Galactose comprises half of the milk sugar lactose and is

thus a major fuel constituent of dairy products. Galactose

and glucose are epimers that differ only in their configura-

tion about C4:

OH H

H OH

H H

α-D-Glucose

OH H

H OH

HO H

α-D-Galactose

The enzymes of glycolysis are specific; they do not recognize

the galactose configuration.An epimerization reaction must

therefore be carried out before galactose enters the gly-

colytic pathway. This reaction takes place after the conver-

sion of galactose to its uridine diphosphate derivative. The

role of UDP–sugars and other nucleotidyl–sugars is dis-

cussed in more detail in Sections 18-2 and 23-3. The entire

pathway converting galactose to a glycolytic intermediate,

which was elucidated by Luis Leloir and is therefore known

as the Leloir pathway, involves four reactions (Fig. 17-36):

1. Galactose is phosphorylated at C1 by ATP in a reac-

tion catalyzed by galactokinase.

2. Galactose-1-phosphate uridylyltransferase transfers

UDP–glucose’s uridylyl group to galactose-1-phosphate to

yield glucose-1-phosphate (G1P) and UDP–galactose by the

reversible cleavage of UDP–glucose’s pyrophosphoryl bond.

Section 17-5. Metabolism of Hexoses Other Than Glucose 631

Figure 17-35 Metabolism of fructose. In muscle (left), the

conversion of fructose to the glycolytic intermediate F6P involves

only one enzyme, hexokinase. In liver (right), seven enzymes

participate in the conversion of fructose to glycolytic

–2

POCH

OPO

–

OPO

–

OPO

–

OPO

–

OPO

–

H OH

H HO

alcohol

dehydrogenase

H OH

H HO

HO H

HOCH

Fructose

Fructose-6-

phosphate

ATP

ADP

fructo-

kinase

H OH

H HO

HO H

Fructose-1-

phosphate

OHCH

HCHO

Fructose-1-phosphate

(open chain)

fructose-1-phosphate

aldolase

C O

Dihydroxy-

acetone

phosphate

C OH

OHCH

Glyceraldehyde

OHCH

Glycerol

ATP

ADP

glycerol

kinase

OHCH

Glycerol-3-phosphate

NADH

glycerol phosphate

dehydrogenase

glycer-

aldehyde

kinase

C OH

OHCH

Glyceraldehyde-3-

phosphate

triose

phosphate

isomerase

ATP

hexokinase

ADP

Glycolysis

NADH NAD

NAD

HO H

Muscle

ATP

ADP

Liver

intermediates: (1) fructokinase, (2) fructose-1-phosphate

aldolase, (3) glyceraldehyde kinase, (4) alcohol dehydrogenase,

(5) glycerol kinase, (6) glycerol phosphate dehydrogenase, and

(7) triose phosphate isomerase.

JWCL281_c17_593-637.qxd 6/3/10 8:38 AM Page 631

3. UDP–galactose-4-epimerase converts UDP–galactose

to UDP–glucose. This enzyme has an associated NAD



which suggests that the reaction involves the sequential

oxidation and reduction of the hexose C4 atom:

4. G1P is converted to the glycolytic intermediate G6P

by the action of phosphoglucomutase (Section 18-1B).

632 Chapter 17. Glycolysis

Figure 17-36 Metabolism of galactose. Four enzymes

participate in the conversion of galactose to the glycolytic

intermediate G6P: (1) galactokinase, (2) galactose-1-phosphate

OH H

HOH

Galactose

ATP

galactokinase

ADP

OH H

HOH

OPO

Galactose-1-phosphate

–

OH H

HOH

OPO

Glucose-1-phosphate (G1P)

phosphoglucomutase

OH H

HOH

Glucose-6-phosphate

(G6P)

–

OHCH

OPO

OH H

HOH

UDP–glucose

–

O Uridine

OH H

HOH

UDP–galactose

–

O Uridine

galactose-1-phosphate

uridylyl transferase

UDP–galactose-

4-epimerase

NAD

Glycolysis

uridylyltransferase, (3) UDP–galactose-4-epimerase, and

(4) phosphoglucomutase.

OH H

H OH

HO H

UDP

UDP–Galactose

OH H

H OH

UDP–Glucose

OHCH

NAD

NADHNADH

OH H

H OH

O UDP

UDP

a. Galactosemia

Galactosemia is a genetic disease characterized by the

inability to convert galactose to glucose. Its symptoms in-

clude failure to thrive, mental retardation, and in some in-

stances death from liver damage. Most cases of galac-

tosemia involve a mutation in the enzyme catalyzing

Reaction 2 of the interconversion, galactose-1-phosphate

uridylyltransferase. This reaction is a double displacement

in which an enzyme His side chain first nucleophilically at-

tacks the -phosphoryl group of UDP–glucose, displacing

G1P and forming a uridylyl–His intermediate:

Galactose-1-phosphate then displaces the uridylyl group

from the enzyme’s His to form UDP–galactose:

A Gln residue forms hydrogen bonds to the uridylyl group’s

phosphoryl oxygens to stabilize the intermediate uridy-

lyl–His. Mutation of this Gln to Arg inactivates the enzyme.

Formation of UDP–galactose from galactose-1-phosphate

UDP–glactose  E–His

Galactose-1-phosphate  E–His-UMP Δ

glucose-1-phosphate  E–His-UMP

UDP–glucose  E–His166 Δ

JWCL281_c17_593-637.qxd 2/26/10 1:38 PM Page 632

is thus prevented, leading to a buildup of toxic metabolic

by-products. For example, the increased galactose concen-

tration in the blood results in a higher galactose concentra-

tion in the lens of the eye where this sugar is reduced to

galactitol.

The presence of this sugar alcohol in the lens eventually

causes cataract formation (clouding of the lens).

Galactosemia is treated by a galactose-free diet. Except

for the mental retardation, this reverses all symptoms of

the disease. The galactosyl units that are essential for the

synthesis of glycoproteins (Section 11-3C) and glycolipids

(Section 12-1D) may be synthesized from glucose by a re-

versal of the epimerase reaction.These syntheses therefore

do not require dietary galactose.

OHCH

HCHO

OHH

Galactitol

C. Mannose

Mannose, a common component of glycoproteins (Section

11-3C), and glucose are C2 epimers:

Mannose enters the glycolytic pathway after its conversion

to F6P via a two-reaction pathway (Fig. 17-37):

1. Hexokinase (Section 17-2A) converts mannose to

mannose-6-phosphate.

2. Phosphomannose isomerase then converts this al-

dose to the ketose F6P. The mechanism of the phospho-

mannose isomerase reaction resembles that catalyzed by

phosphoglucose isomerase (Section 17-2B); it involves an

enediolate intermediate.

OH H

H OH

α-D-Glucose

OH HO

H H

α-D-Mannose

Chapter Summary 633

1 The Glycolytic Pathway Glycolysis is the metabolic

pathway by which most life-forms degrade glucose to two mol-

ecules of pyruvate with the concomitant net generation of two

ATPs. The overall reaction:

occurs in 10 enzymatically catalyzed reactions.

2 The Reactions of Glycolysis In the preparatory stage

of glycolysis, which encompasses its first five reactions, glucose

reacts with two ATPs, in an “energy investment,” to form

fructose-1,6-bisphosphate, which is subsequently converted to

two molecules of glyceraldehyde-3-phosphate. In the second

stage of glycolysis, the “payoff” stage, which comprises its last

five reactions, glyceraldehyde-3-phosphate reacts with NAD

⫹

and P

to form the “high-energy” compound 1,3-bisphospho-

glycerate. This compound reacts in the last four reactions of

2NADH ⫹ 2pyruvate ⫹ 2ATP ⫹ 2H

O ⫹ 4H

⫹

Glucose ⫹ 2NAD

⫹

⫹ 2ADP ⫹ 2P

the pathway with two ADPs to form pyruvate and two ATPs

per molecule. The mechanisms of the 10 glycolytic enzymes

have been elucidated through chemical and kinetic measure-

ments combined with X-ray structural studies. The glycolytic

enzymes exhibit stereospecificity in the reactions that they cat-

alyze. In at least two kinases,phosphoryl transfer from substrate

to water is prevented by substrate-induced conformational

changes that form the active site and exclude water from it.

3 Fermentation: The Anaerobic Fate of Pyruvate The

NAD

⫹

consumed in the formation of 1,3-BPG must be regener-

ated if glycolysis is to continue. In the presence of O

,NAD

⫹

regenerated by oxidative phosphorylation in the mitochondria.

Under anaerobic conditions in muscle, pyruvate is reduced by

NADH, yielding lactate and NAD

⫹

in a reaction catalyzed by

lactate dehydrogenase. In many muscles, particularly during

strenuous activity, the process of homolactic fermentation is a

major free energy source. In anaerobic yeast, NAD

⫹

is regener-

ated by alcoholic fermentation in two reactions.First pyruvate is

CHAPTER SUMMARY

Figure 17-37 Metabolism of mannose. Two enzymes are required to convert mannose to the

glycolytic intermediate F6P: (1) hexokinase and (2) phosphomannose isomerase.

OH HO

H H

Mannose

ATP

hexokinase

ADP

OH HO

H H

Mannose-6-phosphate

phosphomannose

isomerase

OPO

2–

H OH

H HO

HO H

–2

POCH

Fructose-6-phosphate (F6P)

JWCL281_c17_593-637.qxd 6/3/10 8:39 AM Page 633

634 Chapter 17. Glycolysis

decarboxylated to acetaldehyde by pyruvate decarboxylase, an

enzyme that requires thiamine pyrophosphate as a cofactor.

The acetaldehyde is then reduced by NADH to form ethanol

and NAD



in a reaction catalyzed by alcohol dehydrogenase.

4 Metabolic Regulation and Control Metabolic regula-

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

through a pathway is maintained. Metabolic control is the force

exerted on the enzymes of the pathway in response to an exter-

nal signal in order to increase or decrease the flow while main-

taining the steady state to the extent possible. Homeostasis is

the regulation of the steady state. Metabolic flow must be con-

trolled to balance supply with demand and also to maintain

homeostasis. It is possible for more than one enzyme to be rate-

limiting in a metabolic pathway. Metabolic control analysis pro-

vides a framework for the study of metabolic systems in vivo

that share control among more than one enzyme, and it quanti-

tatively describes flux control and homeostasis.The flux control

coefficient measures the sensitivity of the flux to a change in en-

zyme concentration.The elasticity coefficient measures the sen-

sitivity of an enzymatic rate to the change in substrate concen-

tration. Both supply and demand are involved in flux control

and homeostasis. The response of the steady-state concentra-

tion of intermediates to changes in the supply or demand blocks

depends entirely on the elasticity coefficients of the two blocks

at the steady state. When the supply elasticity coefficient is

greater than the demand elasticity coefficient, flux control lies

in the demand portion of the pathway, and vice versa. Home-

ostasis control depends on large elasticity coefficients, whereas

flux control requires a low elasticity coefficient and a high flux

control coefficient. If the demand block controls the flux, the

function of the supply block is to control homeostasis. Feedback

inhibition determines the range of concentration of intermedi-

ates at which there is a steady state. It maintains homeostasis at

physiologically reasonable metabolite concentrations, some-

times far from their equilibrium values.

The flux through a reaction that is close to equilibrium is

very sensitive to changes in substrate concentration. Hence,

the steady-state flux through a metabolic pathway can only be

regulated by a nonequilibrium reaction. Nonequilibrium reac-

tions are controlled by allosteric interactions, substrate cycles,

covalent modification, and genetic (long-term) control mecha-

nisms. In muscle glycolysis, phosphofructokinase (PFK) cat-

alyzes one of the flux-generating steps.Although PFK is inhib-

ited by high concentrations of one of its substrates, ATP, the

10% variation of [ATP] over the range of metabolic activity

has insufficient influence on PFK activity to account for the

observed 100-fold range in glycolytic flux. [AMP] has a 4-fold

variation in response to the 10% variation of [ATP] through

the action of adenylate kinase. Although AMP relieves the

ATP inhibition of PFK, its concentration variation is also in-

sufficient to account for the observed glycolytic flux range.

However, the product of the PFK reaction, fructose-1,6-

bisphosphate, is hydrolyzed to F6P by FBPase, which is inhib-

ited by AMP. The substrate cycle catalyzed by these two en-

zymes confers, at least in principle, the necessary sensitivity of

the glycolytic flux to variations in [AMP]. Substrate cycling is

an important source of nonshivering thermogenesis.

5 Metabolism of Hexoses Other than Glucose Diges-

tion of carbohydrates yields glucose as the primary product.

Other prominent products are fructose, galactose, and man-

nose. These monosaccharides are metabolized through their

conversion to glycolytic intermediates.

General

Cornish-Bowden, A. (Ed.), New Beer in an Old Bottle: Eduard

Buchner and the Growth of Biochemical Knowledge, Universi-

tat de València (1997). [The 1897 paper by Eduard Buchner re-

porting the discovery of cell-free fermentation (in the original

German as well as its English and Spanish translations) to-

gether with a series of essays discussing the historical context of

this discovery and the modern study of multienzyme systems.]

Fersht, A., Structure and Mechanism in Protein Science, Freeman

(1999).

Frey, P.A. and Hegeman, A.D., Enzymatic Reaction Mechanisms,

Oxford University Press (2007).

Fruton, J.S., Molecules and Life: Historical Essays on the Interplay

of Chemistry and Biology, Wiley–Interscience (1974). [In-

cludes a detailed historical account of the elucidation of fer-

mentation.]

Kim, J. and Dang, C.V., Multifaceted roles of glycolytic enzymes,

Trends Biochem. Sci. 30, 142–150 (2005). [Discusses the vari-

ous nonglycolytic functions, mainly regulatory, that glycolytic

enzymes have acquired.]

Saier, M.H., Jr., Enzymes in Metabolic Pathways, Chapter 5,

Harper & Row (1987).

Enzymes of Glycolysis

The Enzymes of Glycolysis: Structure, Activity and Evolution,

Philos. Trans. R. Soc. London Ser. B 293, 1–214 (1981). [A

collection of authoritative discussions on the enzymes of gly-

colysis.]

Allen, S.C. and Muirhead, H., Refined three-dimensional struc-

ture of cat-muscle (M1) pyruvate kinase at a resolution of

2.6 Å, Acta Cryst. D52, 499–504 (1996).

Bennett, W.S., Jr. and Steitz, T.A., Glucose-induced conforma-

tional change in yeast hexokinase, Proc. Natl. Acad. Sci. 75,

4848–4852 (1978).

Bernstein, B.E., Michels, P.A.M., and Hol, W.G.J., Synergistic ef-

fects of substrate-induced conformational changes in phospho-

glycerate activation, Nature 385, 275–278 (1997).

Biesecker, G., Harris, J.I.,Thierry, J.C.,Walker, J.E., and Wonacott,

A.J., Sequence and structure of D-glyceraldehyde-3-phosphate

dehydrogenase from Bacillus stearothermophilus, Nature 266,

328–333 (1977).

Cleland, W.W. and Kreevoy, M.M., Low-barrier hydrogen bonds

and enzymic catalysis, Science 264, 1887–1890 (1994); and

Gerlt, J.A. and Gassman, P.G., Understanding the rates of cer-

tain enzyme-catalyzed reactions: Proton abstraction from car-

bon acids, acyl-transfer reactions, and displacement of phos-

phodiesters, Biochemistry 32, 11943–11952 (1993).

Dalby,A., Dauter, Z., and Littlechild,J.A., Crystal structure of hu-

man muscle aldolase complexed with fructose 1,6-bisphos-

phate: Mechanistic implications, Protein Sci. 8, 291–297 (1999).

Davenport, R.C., Bash, P.A., Seaton, B.A., Karplus, M., Petsko,

G.A., and Ringe,D., Structure of the triosephosphate isomerase–

REFERENCES

JWCL281_c17_593-637.qxd 2/26/10 1:38 PM Page 634

References 635

phosphoglycohydroxamate complex:An analogue of the inter-

mediate on the reaction pathway, Biochemistry 30, 5821–5826

(1991); and Lolis, E. and Petsko, G.A., Crystallographic analy-

sis of the complex between triosephosphate isomerase and

2-phosphoglycolate at 2.5 Å resolution: Implications for catal-

ysis, Biochemistry 29, 6619–6625 (1990).

Evans, P.R. and Hudson, P.J., Structure and control of phospho-

fructokinase from Bacillus stearothermophilus, Nature 279,

500–504 (1979).

Gefflaut,T., Blonski, C.,Perie, J., and Willson,M., Class I aldolases:

Substrate specificity, mechanism, inhibitors and structural

aspects, Prog. Biophys. Mol. Biol. 63, 301–340 (1995).

Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A., and

Hunter, W.N., The crystal structure of Escherichia coli class II

fructose-1,6-bisphosphate aldolase in complex with phospho-

glycohydroxamate reveals details of mechanism and speci-

ficity, J. Mol. Biol. 287, 383–394 (1999).

Harlos, K., Vas, M., and Blake, C.C.F., Crystal structure of the bi-

nary complex of pig muscle phosphoglycerate kinase and its

substrate 3-phospho-

D-glycerate, Proteins 12, 133–144 (1992).

Jedrzejas, M.J., Structure, function, and evolution of phosphoglyc-

erate mutase: Comparison with fructose-2,6-bisphosphatase,

acid phosphatase, and alkaline phosphatase, Prog. Biophys.

Mol. Biol. 73, 263–287 (2000).

Jeffrey, C.J., Bahnson, B.J., Chien, W., Ringe, D., and Petsko, G.A.,

Crystal structure of rabbit phosphoglucose isomerase, a glycolytic

enzyme that moonlights as neuroleukin, autocrine motility factor,

and differentiation mediator, Biochemistry 39, 955–964 (2000).

Jogl, G., Rozovsky, S., McDermott, A.E., and Tong, L., Optimal

alignment for enzymatic proton transfer: Structure of the

Michaelis complex of triosphosphate isomerase at 1.2-Å reso-

lution, Proc. Natl.Acad. Sci. 100, 50–55 (2003).

Joseph, D., Petsko, G.A., and Karplus, M., Anatomy of a confor-

mational change: Hinged “lid” motion of the triosephosphate

isomerase loop, Science 249, 1425–1428 (1990).

Knowles, J.R., Enzyme catalysis: Not different, just better, Nature

350, 121–124 (1991). [A lucid discussion of TIM’s catalytic

mechanism.]

Kuby, S.A. (Ed.), A Study of Enzymes,Vol. II, CRC Press (1991).

[Chapters 17, 18, 19, and 20 discuss the mechanisms of adeny-

late kinase, PFK, PGI and TIM, and aldolase, respectively.

Chapter 4 discusses thiamine-dependent reaction mechanisms.]

Kuser, P., Cupri, F., Bleicher, L., and Polikarpov, I., Crystal struc-

ture of yeast hexokinase PI in complex with glucose: A classi-

cal “induced fit” example revised, Proteins 72, 731–740 (2008).

Marsh, J.J. and Lebherz, H.G., Fructose-bisphosphate aldolases:An

evolutionary history, Trends Biochem. Sci. 17, 110–113 (1992).

Maurer, P.J. and Nowak,T., Fluoride inhibition of yeast enolase. 1.

Formation of ligand complexes, Biochemistry 20, 6894–6900

(1981); and Nowak, T. and Maurer, P.J., Fluoride inhibition of

yeast enolase. 2. Structural and kinetic properties of ligand

complexes determined by nuclear relaxation rate studies, Bio-

chemistry 20, 6901–6911 (1981).

Morris,A.J. and Tolan,D.R., Lysine-146 of rabbit muscle aldolase is

essential for cleavage and condensation of the C3-C4 bond of

fructose 1,6-bis(phosphate), Biochemistry 33, 12291–12297

(1994); and Site-directed mutagenesis identifies aspartate 33 as

a previously unidentified critical residue in the catalytic mecha-

nism of rabbit aldolase A, J. Biol. Chem. 268, 1095–1100 (1993).

Muirhead, H. and Watson, H. Glycolytic enzymes: From hexose to

pyruvate, Curr. Opin. Struct. Biol. 2, 870–876 (1992).

Reed, G.H., Poyner, R.R., Larsen, T.M., Wedekind, J.E., and

Rayment, I., Structural and mechanistic studies on enolase,

Curr. Opin. Struct. Biol. 6, 736–743 (1996).

Seeholzer, S.H., Phosphoglucose isomerase: A ketol isomerase

with aldol C2-epimerase activity, Proc. Natl. Acad. Sci. 90,

1237–1241 (1993).

Williams, J.C.and McDermott,A.E., Dynamics of the flexible loop

of triosephosphate isomerase: The loop motion is not ligand-

gated, Biochemistry 34, 8309–8319 (1995).

Enzymes of Anaerobic Fermentation

Boyer, P.D. (Ed.), The Enzymes (3rd ed.),Vol. 11,Academic Press

(1975). [Contains authoritative reviews on alcohol dehydroge-

nase, lactate dehydrogenase, and the evolutionary and struc-

tural relationships among the dehydrogenases.]

Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., and

Jordan, F., Catalytic centers in the thiamin diphosphate de-

pendent enzyme pyruvate decarboxylase at 2.4-Å resolution,

Biochemistry 32, 6165–6170 (1993).

Golbik, R., Neef, H., Hubner, G., Konig, S., Seliger, B., Meshalkina,

L., Kochetov, G.A., and Schellenberger, A., Function of the

aminopyridine part in thiamine pyrophosphate enzymes,

Bioinorg. Chem. 19, 10–17 (1991).

Park, J.H., Brown, R.L., Park, C.R., Cohn, M., and Chance, B., En-

ergy metabolism in the untrained muscle of elite runners as

observed by

P magnetic resonance spectroscopy: Evidence

suggesting a genetic endowment for endurance exercise. Proc.

Natl.Acad. Sci. 85, 8780–8785 (1988).

Control of Metabolic Flux

Crabtree, B. and Newsholme, E.A., A systematic approach to de-

scribing and analyzing metabolic control systems, Trends

Biochem. Sci. 12, 4–12 (1987).

Fell, D.A., Metabolic control analysis:A survey of its theoretical and

experimental development, Biochem. J. 286, 313–330 (1992).

Fell, D., Understanding the Control of Metabolism, Portland Press

(1997).

Hofmeyr, J.-H.S. and Cornish-Bowden,A., Regulating the cellular

economy of supply and demand, FEBS Lett. 476, 47–51 (2000).

Kacser, H. and Burns, J.A. (with additional comments by Kacser,

H. and Fell, D.A.),The control of flux, Biochem. Soc.Trans. 23,

341–366 (1995).

Kacser, H. and Porteous, J.W., Control of metabolism:What do we

have to measure? Trends Biochem. Sci. 12, 5–14 (1987).

Lardy, H. and Schrago, E., Biochemical aspects of obesity, Annu.

Rev. Biochem. 59, 689–710 (1990).

Newsholme, E.A., Challiss, R.A.J., and Crabtree, B., Substrate cy-

cles: their role in improving sensitivity in metabolic control,

Trends Biochem. Sci. 9, 277–280 (1984).

Perutz, M.F., Mechanism of cooperativity and allosteric regulation

in proteins, Q. Rev. Biophys. 22, 139–236 (1989). [Section 6 dis-

cusses PFK.]

Schaaf, I., Heinisch, J., and Zimmermann, K., Overproduction of

glycolytic enzymes in yeast, Yeast 5, 285–290 (1989).

Schirmer, T. and Evans, P.R., Structural basis of the allosteric

behaviour of phosphofructokinase, Nature 343, 140–145 (1990).

Walsh, K. and Koshland, D.E., Jr., Characterization of rate-

controlling steps in vivo by use of an adjustable expression

vector, Proc. Natl. Acad. Sci. 82, 3577–3581 (1985).

Metabolism of Hexoses Other Than Glucose

Frey, P.A.,The Leloir pathway:A mechanistic imperative for three

enzymes to change the stereochemical configuration of a sin-

gle carbon in galactose, FASEB J. 10, 461–470 (1996).

Valle, D. (Ed.), The Metabolic & Molecular Bases of Inherited Dis-

ease, http://www.ommid.com. [Chapters 70 and 72 discuss

fructose and galactose metabolism and their genetic disorders.]

JWCL281_c17_593-637.qxd 6/30/10 11:02 AM Page 635

636 Chapter 17. Glycolysis

1. Write out the reactions of the glycolytic pathway from glu-

cose to lactate using structural formulas for all intermediates.

Learn the names of these intermediates and the enzymes that cat-

alyze the reactions.

2. ⌬G°¿ for the aldolase reaction is ⫹22.8 kJ ⴢ mol

⫺1

. In the cell,

at 37°C, the mass action ratio [DHAP]/[GAP] ⫽ 5.5. Calculate the

equilibrium ratio of [FBP]/[GAP] when [GAP] is (a) 2 ⫻ 10

⫺5

and (b) 10

⫺3

3. The pH dependence of the rate of the triose phosphate iso-

merase (TIM) reaction has characteristic pK’s of 6.5 and 9.5.His 95,

a catalytically essential residue, has been shown to have a pK of 4.5.

Why doesn’t the pH rate curve indicate the existence of this pK?

4. Arsenate , a structural analog of phosphate, can

act as a substrate for any reaction in which phosphate is a sub-

strate. Arsenate esters, unlike phosphate esters, are kinetically as

well as thermodynamically unstable and hydrolyze almost instan-

taneously. Write a balanced overall equation for conversion of

glucose to pyruvate in the presence of ATP, ADP, NAD

⫹

, and ei-

ther (a) phosphate or (b) arsenate. (c) Why is arsenate a poison?

5. When glucose is degraded anaerobically via glycolysis there is

no overall oxidation or reduction of the substrate.The fermentation

reaction is therefore said to be “balanced.”The free energy required

for ATP formation is nevertheless obtained from favorable electron-

transfer reactions. Which metabolic intermediate is the electron

donor and which is the electron acceptor when glucose is degraded

by a balanced glycolytic fermentation:(a) in muscle and (b) in yeast?

6. In which carbon atoms of pyruvate would radioactivity be

found if glucose metabolized by the glycolytic pathway were la-

beled with

C at: (a) C1 and (b) C4? (Note: Assume that triose

phosphate isomerase is able to equilibrate dihydroxyacetone

phosphate and glyceraldehyde-3-phosphate.)

*7. The following reaction is catalyzed by an enzyme very sim-

ilar to Class I aldolases:

HO H

OPO

2⫺

OPO

2⫺

Fructose-6-

phosphate

Erythrose-4-

phosphate

OPO

2⫺

CHOH

OPO

2⫺

Sedoheptulose-7-

phosphate

Glyceraldehyde-3-

phosphate

⫹

transaldolase

(AsO

3⫺

)

Write a plausible mechanism for this reaction using curved arrows

to indicate the electron flow.

8. The half-reactions involved in the LDH reaction and their

standard reduction potentials are

Calculate ⌬G for the reaction at the biochemical standard state

under the following conditions:

(a) [lactate]/[pyruvate] ⫽ 1; [NAD

⫹

]/[NADH] ⫽ 1

(b) [lactate]/[pyruvate] ⫽ 160; [NAD

⫹

]/[NADH] ⫽ 160

⫹

]/[NADH] ⫽ 1000

(d) Under what conditions will the reaction spontaneously

favor NADH oxidation?

(e) In order for the free energy change of the glyceraldehyde-

3-phosphate dehydrogenase reaction to favor glycolysis,

the [NAD

⫹

]/[NADH] ratio must be maintained close to

. Under anaerobic conditions in mammalian muscle,

lactate dehydrogenase performs this function. How high

can the [lactate]/[pyruvate] ratio become in muscle cells

before the LDH-catalyzed reaction ceases to be favorable

in the direction of NAD

⫹

production while maintaining

the foregoing [NAD

⫹

]/[NADH] ratio constant?

9. Although it is not the primary flux-control point for glycol-

ysis, pyruvate kinase is subject to allosteric regulation. (a) What is

the metabolic importance of regulating flux through the pyruvate

kinase reaction? (b) What is the advantage of activating pyruvate

kinase with fructose-1,6-bisphosphate?

*10. Based on the involvement of thiamine pyrophosphate

(TPP) in the pyruvate decarboxylase reaction, which of the fol-

lowing reactions, if any, might be expected to utilize TPP as a

cofactor?

Write hypothetical mechanisms for each reaction showing where

TPP is involved or why it is unnecessary.

11. The glycolytic pathway for glucose breakdown is almost

universal. Some bacteria, however, utilize an alternate route

called the Entner–Doudoroff pathway (Fig. 17-38). Like the gly-

colytic pathway in yeast, the final product is ethanol. (a) Write bal-

anced equations for the conversion of glucose to ethanol and CO

via the Entner–Doudoroff pathway and the yeast alcoholic fer-

mentation. (b) Infer from your stoichiometries why the glycolytic

pathway rather than the Entner–Doudoroff pathway is almost

universal.

⫺

C H

CCO

⫺

⫹

HOCO

⫺

⫹

(a)

(b)

NAD

⫹

⫹ 2H

⫹

⫹ 2e

⫺

NADH ⫹ H

⫹

e°¿ ⫽⫺0.315 V

Pyruvate ⫹ 2H

⫹

⫹ 2e

⫺

lactate

e°¿ ⫽⫺0.185 V

PROBLEMS

JWCL281_c17_593-637.qxd 6/30/10 2:19 PM Page 636

*12. The hydrolysis of ATP to ADP in the cell results in a con-

comitant change in [AMP] as mediated by adenylate kinase. (a)

Assuming that [ATP]  [AMP] and that the total adenine nu-

cleotide concentration in the cell, A

 [AMP]  [ADP]  [ATP],

Problems 637

is constant, derive an expression for [AMP] in terms of [ATP] and

. (b) Assuming an initial [ATP]/[ADP] of 10 and A

 5 mM,

calculate the ratio of the final to initial values of [AMP] on a 10%

decrease of [ATP].

Figure 17-38 Entner–Doudoroff pathway for glucose breakdown.

CHOH

2

2

O P



O P



C OHH

2ATP

C O



O P

glucokinase

ATP ADP

glucose-6-

phosphate

dehydrogenase

NADH

6-phospho-

gluconate

dehydrase

(analogous

to enolase)

Glucose Glucose-6-

phosphate

(G6P)

6-Phospho-

gluconate

2-Keto-3-deoxy-

6-phosphogluconate

(KDPG)

321



NAD



NAD



NADH

KDPG-

aldolase

Pyruvate

Glyceraldehyde-3-

phosphate (GAP)

same reactions as

in glycolysis and

alcoholic fermentation

Ethanol

2ADP  P

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

638

CHAPTER 18

Glycogen Metabolism

1 Glycogen Breakdown

A. Glycogen Phosphorylase

B. Phosphoglucomutase

C. Glycogen Debranching Enzyme

D. Thermodynamics of Glycogen Metabolism: The Need for

Separate Pathways of Synthesis and Breakdown

2 Glycogen Synthesis

A. UDP–Glucose Pyrophosphorylase

B. Glycogen Synthase

C. Glycogen Branching Enzyme

3 Control of Glycogen Metabolism

A. Direct Allosteric Control of Glycogen Phosphorylase and

Glycogen Synthase

B. Covalent Modification of Enzymes by Cyclic Cascades:

Effector “Signal” Amplification

C. Glycogen Phosphorylase Bicyclic Cascade

D. Glycogen Synthase Bicyclic Cascade

E. Integration of Glycogen Metabolism Control Mechanisms

F. Maintenance of Blood Glucose Levels

G. Response to Stress

4 Glycogen Storage Diseases

Everything should be made as simple as possible, but not

simpler.

Albert Einstein

Glucose, a major metabolic fuel source,is degraded via gly-

colysis to produce ATP (Chapter 17). Higher organisms

protect themselves from potential fuel shortage by poly-

merizing excess glucose for storage as high molecular mass

glucans (glucose polysaccharides) that may be readily mo-

bilized in times of metabolic need. In plants, this glucose

storage substance is starch, a mixture of the (1 S 4)-linked

glucan -amylose (Fig. 11-18) and amylopectin, which dif-

fers from -amylose by the presence of (1 S 6) branches

every 24 to 30 residues (Fig. 11-19). In animals, the storage

glucan is glycogen (Fig. 18-1), which differs from amy-

lopectin only in that its branches occur every 8 to 14

residues. Glycogen occurs in 100- to 400-Å-diameter cyto-

plasmic granules (Figs. 11-20 and 18-1c), which contain up

to 120,000 glucose units. They are especially prominent in

the cells that make the greatest use of glycogen, muscle

(maximally 1–2% glycogen by weight) and liver cells (max-

imally 10% glycogen by weight, an ⬃12-h energy supply

for the body). Glycogen granules also contain the enzymes

that catalyze glycogen synthesis and degradation as well as

some of the enzymes that regulate these processes.

As we shall see in this chapter, glycogen’s glucose units

are mobilized by their sequential removal from the glucan

chains’ nonreducing ends (ends lacking a C1 OH group).

Glycogen’s highly branched structure is therefore physio-

logically significant: It permits glycogen’s rapid degradation

through the simultaneous release of the glucose units at the

end of every branch.

Why does the body go to such metabolic effort to use

glycogen for energy storage when fat, which is far more

abundant in the body, seemingly serves the same purpose?

The answer is threefold:

1. Muscles cannot mobilize fat as rapidly as they can

glycogen.

2. The fatty acid residues of fat cannot be metabolized

anaerobically (Section 25-2).

3. Animals cannot convert fatty acids to glucose (Sec-

tion 23-1), so fat metabolism alone cannot adequately

maintain essential blood glucose levels (Section 18-3F).

As with all metabolic processes, there are several levels on

which glycogen metabolism may be understood.We shall ex-

amine this process in order to understand the pathway’s ther-

modynamics and the reaction mechanisms of its individual

steps but will emphasize the mechanisms by which glycogen

synthesis and breakdown rates are controlled.We began our

consideration of metabolic control mechanisms in Section

17-4 with a discussion of the role of allosteric interactions and

substrate cycles in the regulation of glycolysis. Glycogen me-

tabolism’s more complex control systems provide us with ex-

amples of several additional control processes:covalent mod-

ification of enzymes and enzyme cascades. In addition, we

shall consider glycogen metabolism as a model for the role of

hormones in the overall regulatory process.We end the chap-

ter by discussing the consequences of genetic defects in vari-

ous enzymes of glycogen metabolism.

1 GLYCOGEN BREAKDOWN

Liver and muscle are the two major storage tissues for

glycogen. In muscle,the need for ATP results in the conver-

sion of glycogen to glucose-6-phosphate (G6P) for entry

into glycolysis. In liver, low blood glucose concentration

triggers glycogen breakdown to G6P, which in this case is

JWCL281_c18_638-670.qxd 2/26/10 2:24 PM Page 638

hydrolyzed to glucose and released into the bloodstream to

reverse this situation.

Glycogen breakdown requires the actions of three

enzymes:

1. Glycogen phosphorylase (or simply phosphorylase)

catalyzes glycogen phosphorolysis (bond cleavage by the

substitution of a phosphate group) to yield glucose-1-

phosphate (G1P).

(n residues) (n  1 residues)

This enzyme will only release a glucose unit that is at least

five units from a branch point.

2. Glycogen debranching enzyme removes glycogen’s

branches, thereby permitting the glycogen phosphorylase re-

action to go to completion. It also hydrolyzes (1 S 6)-linked

glucosyl units to yield glucose. Consequently, ⬃92% of glyco-

gen’s glucose residues are converted to G1P. The remaining

⬃8%, those at the branch points, are converted to glucose.

3. Phosphoglucomutase converts G1P to G6P, which, as

we have seen (Section 17-2A),is also formed in the first step

of glycolysis through the action of either hexokinase or glu-

cokinase. G6P can either continue along the glycolytic path-

way (as in muscle) or be hydrolyzed to glucose (as in liver).

In this section, we discuss the structures and mechanisms of

action of these three enzymes.

A. Glycogen Phosphorylase

Glycogen phosphorylase is a dimer of identical 842-residue

(97-kD) subunits that catalyzes the controlling step in glyco-

gen breakdown.It is regulated both by allosteric interactions

and by covalent modification. The enzyme-catalyzed modifi-

cation/demodification process yields two forms of phospho-

rylase: phosphorylase a, which has a phosphoryl group ester-

ified to Ser 14 in each of its subunits, and phosphorylase b,

which lacks these phosphoryl groups. Phosphorylase’s al-

losteric inhibitors,ATP,G6P, and glucose,and its allosteric ac-

tivator, AMP (to name only the enzyme’s most prominent

Glycogen  P

glycogen

 G1P

Section 18-1. Glycogen Breakdown 639

Nonreducing

ends

Branch

point

(a)

α(1 6) linkage

linkage

α(1 4)

Reducing

end

OH H

HHH

HOH

OH H

HOH

OH H

HOH

OH H

HHH

HOH

OH H

HOH

OH H

HOH

OH H

HOH

OH H

HOH

Branch

point

Reducing

end

Nonreducing

end

(b)

Figure 18-1 Structure of glycogen. (a) Molecular formula. In

the actual molecule the chains are much longer than shown.

(b) Schematic diagram illustrating its branched structure. Branch

points in the actual molecule are separated by 8 to 14 glucosyl

units. Note that the molecule, no matter how big, has but one

reducing end. (c) Electron micrograph of a glycogen granule

from rat skeletal muscle. Each granule () consists of several

spherical glycogen molecules () and its associated proteins.

Elsevier Science. Used with permission.]

(c)

JWCL281_c18_638-670.qxd 6/3/10 1:47 PM Page 639

effectors), interact differently with the phospho- and dephos-

phoenzymes, resulting in an extremely sensitive control

process. We study this control process in Section 18-3C.

a. Structural Domains and Binding Sites

The high-resolution X-ray structures of phosphorylase a

and phosphorylase b were determined by Robert Fletterick

and Louise Johnson, respectively.The structure of phospho-

rylase b, despite its lack of a Ser-linked phosphate, is very

similar to that of phosphorylase a (Fig. 18-2). Both struc-

tures have two domains, an N-terminal domain (residues

1–484; among the largest known domains), and a C-termi-

nal domain (residues 485–842). The N-terminal domain is

further divided into an interface subdomain (residues

1–315), which includes the covalent modification site (Ser

14), the allosteric effector site, and all the intersubunit con-

tacts in the dimer; and a glycogen-binding subdomain

(residues 316–484), which contains the “glycogen storage

site”(see below).The catalytic site is located at the center of

the subunit where these two subdomains come together

with the C-terminal domain. In Section 18-3 we discuss the

allosteric behavior of glycogen phosphorylase and the con-

formational differences between phosphorylases a and b.

Glycogen forms a left-handed helix with 6.5 glucose

residues per turn, similar to ␣-amylose (Fig. 11-18b). An

⬃30-Å-long crevice on the surface of the phosphorylase

monomer that has the same radius of curvature as glycogen

connects the glycogen storage site, which binds glycogen, to

the active site, which phosphorylizes it. Since this crevice

can accommodate four or five sugar residues in a chain but

is too narrow to admit branched oligosaccharides, it pro-

vides a clear physical rationale for the inability of phospho-

rylase to cleave glycosyl residues closer than five units from

a branch point. Presumably the glycogen storage site in-

creases the catalytic efficiency of phosphorylase by permit-

ting it to phosphorylize many glucose residues on the same

glycogen particle without having to dissociate and reassoci-

ate completely between catalytic cycles.

b. Pyridoxal Phosphate Is an Essential Cofactor

for Phosphorylase

Phosphorylase contains pyridoxal-5¿¿ -phosphate (PLP)

and requires it for activity. This vitamin B

derivative is co-

valently linked to phosphorylase via a Schiff base to Lys

679. PLP is similarly linked to a variety of enzymes in-

volved in amino acid metabolism, where it is an essential

cofactor in transamination reactions (Section 26-1Aa).The

–

PLP covalently bound to

phosphorylase via a

Schiff base to Lys 679

(CH

)

–

Phosphorylase

Lys 679

Pyridoxal-5ⴕ-phosphate (PLP)

–

640 Chapter 18. Glycogen Metabolism

Figure 18-2 X-ray structure of rabbit muscle glycogen

phosphorylase a. The homodimeric enzyme is drawn in ribbon

form and viewed along its 2-fold axis. Each subunit consists of an

N-terminal domain, which is subdivided into an interface

subdomain (residues 1–315) and a glycogen-binding subdomain

(residues 316–484), and a C-terminal domain (residues 485–842).

The enzyme’s several ligands are drawn in stick form colored

according to type with N blue, O red, P orange, and C atoms as

indicated.The active site is marked by a bound glucose molecule

(C yellow). Pyridoxal phosphate (PLP) is covalently linked to the

side chain of Lys 678 in the C-terminal domain (C magenta). In

addition, the enzyme binds its allosteric effector AMP (C cyan)

and maltoheptaose (C green), an ␣(1 S 4)-linked glucose

heptamer, which is bound in the enzyme’s glycogen storage site.

Ser 14-P, the phosphoryl group on Ser 14, is drawn in space-filling

form. [X-ray structure coordinates courtesy of Stephen Sprang,

University of Texas Southwest Medical Center.]

See

Kinemage Exercise 14-1

JWCL281_c18_638-670.qxd 6/3/10 1:47 PM Page 640