Voet D., Voet Ju.G. Biochemistry

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proteases) and pharmacokinetics (they do not readily pass

through cell membranes.). Consequently, therapeutically

effective HIV-1 protease inhibitors must be peptidomimet-

ics (peptide mimics), substances that sterically and perhaps

physically, but not chemically, resemble polypeptides. The

use of peptidomimetics also permits conformational con-

straints to be imposed on a drug candidate that would not

be present in the corresponding polypeptide.

The FDA has approved ten HIV-1 protease inhibitors

(Fig. 15-41), the first of which, saquinavir, was sanctioned in

late 1995. These peptidomimetics have IC

’s against HIV

in culture ranging from 2 to 60 nM but have little or no ac-

tivity against human aspartic proteases (K

’s  10 M).

They are the first drugs to clearly prolong the lives of AIDS

victims. Their development, in each case, was a complex it-

erative process that required the design, synthesis, and

evaluation of numerous related compounds. In several

cases, these investigations capitalized on the wealth of ex-

perience gained in developing peptidomimetic inhibitors

of the aspartic protease renin and in the resulting stock-

piles of these compounds.

All the FDA-approved HIV-1 protease inhibitors ini-

tially cause a rapid and profound decline in a patient’s

plasma HIV load, which is often paralleled by immune sys-

tem recovery. However, as we saw with reverse transcrip-

tase inhibitors, mutant forms of the protease that are resist-

ant to the inhibitor being used arise, usually within 4 to 12

weeks. Moreover, such a mutant protease is likely to be re-

sistant to other HIV-1 protease inhibitors, because all of the

HIV-1 protease inhibitors are targeted to the same binding

site. This has led to the use of combination therapies in

which an HIV-1 protease inhibitor is administered together

with one, or more often, two inhibitors of other viral

processes (inhibitors of reverse transcriptase, integrase, and

viral entry into host cells). This is because any virus that

gains resistance to one drug in a regimen will be suppressed

by the other drug(s) in that regimen. In addition, the HIV-1

protease inhibitor ritonavir has been shown to be a potent

inhibitor of the cytochrome P450 isoforms (CYP3A4,5,7)

that metabolize other protease inhibitors and hence is usu-

ally prescribed in low dosage as an adjunct to another pro-

tease inhibitor to improve the latter’s pharmacokinetics.

The plasma virus levels in many patients who were placed

on combination therapy rapidly became undetectable and

have remained so for several years. This, however, does not

constitute a cure: If drug therapy is interrupted, the virus will

reappear in the plasma because certain tissues in the body

harbor latent viruses that are unaffected by and/or inaccessi-

ble to drug therapy. Thus, the presently available anti-HIV

medications must be taken for a lifetime.

Current anti-HIV therapies are by no means ideal. To

maximize their oral bioavailability, some of the different

drugs must be taken well before or after a meal but others

must be taken with a meal. To minimize the probability of

resistant forms of HIV arising, the bioavailability of each

drug must be maintained at a certain minimum level and

hence each drug must be taken on a rigid schedule. More-

over, these drugs have significant side effects, mainly

fatigue, nausea, diarrhea, tingling and numbness with riton-

avir, and kidney stones with indinavir. Consequently, nu-

merous AIDS patients fail to take their medications prop-

erly, which greatly increases the likelihood that they will

develop resistance to these drugs and infect others with

drug-resistant viruses. Finally, HIV-1 protease inhibitors,

being complex molecules, are difficult to synthesize and

therefore are relatively expensive, so that in the developing

countries in which AIDS is most prevalent, governments

and most individuals cannot afford to purchase these

drugs, even if they were to be supplied at cost. It is there-

fore important that anti-HIV therapies be developed that

are easy for patients to comply with, are inexpensive, and

ideally, will totally eliminate an HIV infection.

Chapter Summary 551

1 Catalytic Mechanisms Most enzymatic mechanisms of

catalysis have ample precedent in organic catalytic reactions.

Acid- and base-catalyzed reactions occur, respectively, through

the donation or abstraction of a proton to or from a reactant

so as to stabilize the reaction’s transition state complex.

Enzymes often employ ionizable amino acid side chains as

general acid–base catalysts. Covalent catalysis involves nucle-

ophilic attack of the catalyst on the substrate to transiently

form a covalent bond followed by the electrophilic stabiliza-

tion of a developing negative charge in the reaction’s transi-

tion state. Various protein side chains as well as certain coen-

zymes can act as covalent catalysts. Metal ions, which are

common enzymatic components, catalyze reactions by stabi-

lizing developing negative charges in a manner resembling

general acid catalysis. Metal ion–bound water molecules are

potent sources of OH



ions at neutral pH’s. Metal ions also fa-

cilitate enzymatic reactions through the charge shielding of

bound substrates. The arrangement of charged groups about

an enzymatic active site of low dielectric constant in a manner

that stabilizes the transition state complex results in the elec-

trostatic catalysis of the enzymatic reaction. Enzymes catalyze

reactions by bringing their substrates into close proximity in

reactive orientations. The enzymatic binding of the substrates

in a bimolecular reaction arrests their relative motions result-

ing in a rate enhancement.The preferential enzymatic binding

of the transition state of a catalyzed reaction over the sub-

strate is an important rate enhancement mechanism. Transi-

tion state analogs are potent competitive inhibitors because

they bind to the enzyme more tightly than does the correspon-

ding substrate.

2 Lysozyme Lysozyme catalyzes the hydrolysis of

(1 S 4)-linked poly(NAG–NAM), the bacterial cell wall

polysaccharide, as well as that of poly(NAG). Lysozyme

binds a hexasaccharide so as to distort its D-ring toward the

CHAPTER SUMMARY

JWCL281_c15_506-556.qxd 2/19/10 9:28 PM Page 551

half-chair conformation of the planar oxonium ion transition

state. This is followed by cleavage of the bond be-

tween the D- and E-rings as promoted by proton donation

from Glu 35. The resulting oxonium ion transition state is

electrostatically stabilized by the nearby carboxyl group of

Asp 52, which then forms a covalent bond with C1.The E-ring

leaving group is subsequently replaced by water, which in

what is essentially a reversal of the previous reaction se-

quence, attacks C1 yielding the reaction’s second product and

regenerating the enzyme. The roles of Glu 35 and Asp 52 in

lysozyme catalysis have been verified through mutagenesis

studies. Structural and binding studies indicate that strain is of

major catalytic importance in the lysozyme mechanism. Mass

spectrometry and X-ray studies have shown that the lysozyme

reaction proceeds via a covalent glycosyl–enzyme intermedi-

ate involving Asp 52 rather than by the noncovalently bound

oxonium ion intermediate postulated by the original Phillips

mechanism.

3 Serine Proteases Serine proteases constitute a wide-

spread class of proteolytic enzymes that are characterized by

the possession of a reactive Ser residue. The pancreatically

synthesized digestive enzymes trypsin, chymotrypsin, and

elastase are sequentially and structurally related but have

different side chain specificities for their substrates. All have

the same catalytic triad,Asp 102, His 57, and Ser 195, at their

active sites. The differing side chain specificities of trypsin

and chymotrypsin depend in a complex way on the structures

of the loops that connect the walls of the specificity pocket,

as well as on the charge of the side chain at the base of the

specificity pocket. Subtilisin, serine carboxypeptidase II, and

ClpP are unrelated serine proteases that have essentially the

same active site geometry as do the pancreatic enzymes.

Catalysis in serine proteases is initiated by the nucle-

ophilic attack of the active Ser on the carbonyl carbon atom

of the scissile peptide to form the tetrahedral intermediate, a

process that may be facilitated by the formation of a low-

barrier hydrogen bond between Asp 102 and His 57. The

tetrahedral intermediate, which is stabilized by its preferen-

tial binding to the enzyme’s active site, then decomposes to

the acyl–enzyme intermediate under the impetus of proton

donation from the Asp 102-polarized His 57. After the re-

placement of the leaving group by solvent H

O, the catalytic

process is reversed to yield the second product and the re-

generated enzyme. The active Ser is not unusually reactive

but is ideally situated to nucleophilically attack the activated

scissile peptide. The X-ray structure of the trypsin–BPTI

complex indicates the existence of the tetrahedral intermedi-

ate, whereas X-ray structures of a complex of elastase with

the heptapeptide BCM7 have visualized both the acyl–enzyme

intermediate and the tetrahedral intermediate. Enzyme–sub-

strate complexes have free energy landscapes that facilitate

the formation of the transition state.

The pancreatic serine proteases are synthesized as zymo-

gens to prevent pancreatic self-digestion. Trypsinogen is acti-

vated by a single proteolytic cleavage by enteropeptidase. The

resulting trypsin similarly activates trypsinogen as well as chy-

motrypsinogen, proelastase,and other pancreatic digestive en-

zymes. Trypsinogen’s catalytic triad is structurally intact. The

C1¬O1

zymogen’s low catalytic activity arises from a distortion of its

specificity pocket and oxyanion hole, so that it is unable to

productively bind substrate or preferentially bind the catalytic

reaction’s transition state.

4 Drug Design Drugs act by binding to and thereby mod-

ifying the functions of receptors. Many promising drug candi-

dates, which are known as lead compounds, have been found

by methods in which a large number of compounds are tested

for drug efficacy in an assay that is a suitable surrogate of the

disease/condition under consideration. Lead compounds are

then chemically manipulated in the search for compounds

with improved drug efficacy. Structure–activity relationships

(SARs) and quantitative structure–activity relationships

(QSARs) are useful tools in this endeavor. Structure-based

drug design uses the X-ray and NMR structures of drug candi-

dates in complex with their target proteins, together with a va-

riety of molecular modeling tools, to guide the search for im-

proved drug candidates. However,the advent of combinatorial

chemistry, fragment-based lead discovery, and high-throughput

screening procedures has extended the “make-many-compounds-

and-see-what-they-do” approaches to drug discovery.

In order to reach their target receptors, drugs must have

favorable pharmacokinetics, that is, they must readily traverse

numerous physical barriers in the body, avoid chemical trans-

formation by enzymes, and not be excreted too rapidly. Most

useful drugs are neither too lipophilic nor too hydrophilic so

that they can both gain access to the necessary membranes

and pass through them. Drug toxicity, dosage, efficacy, and the

nature of rare adverse reactions are determined through ex-

tensive and carefully designed clinical trials. Most drugs are

metabolically cleared through oxidative hydroxylation by one

of the numerous cytochrome P450 isozymes. This permits the

hydroxylated drugs to be enzymatically conjugated to polar

groups such as glucuronic acid and glycine, which increases

their rates of excretion by the kidneys. Drug–drug interactions

are frequently mediated by cytochromes P450.Polymorphisms

among cytochromes P450 are often responsible for the varia-

tions among individuals in their response to a given drug, in-

cluding adverse reactions.

The formulation of HIV-1 protease inhibitors to control

HIV infections is one of the major triumphs of modern drug

discovery methods. HIV are retroviruses that attack specific

immune system cells and thereby degrade the immune system

over a period of several years to the point that it is no longer

able defend against opportunistic infections. HIV-1 protease

functions to cleave the polyproteins in immature HIV-1 viri-

ons that have budded out from a host cell, thus generating the

mature, infectious form. HIV-1 protease is an aspartic pro-

tease that, as do eukaryotic aspartic proteases such as pepsin,

uses its two active site Asp residues to activate its bound lytic

water molecule as the nucleophile that attacks and thereby

cleaves specific peptide bonds in the substrate polyprotein.

All of the FDA-approved peptidomimetic inhibitors of HIV-1

protease cause a rapid and profound decrease in plasma HIV

levels, although they do not entirely eliminate the virus. They

are used in combination with inhibitors of other viral

processes to minimize the ability of the rapidly mutating HIV

to evolve drug-resistant forms.

552 Chapter 15. Enzymatic Catalysis

JWCL281_c15_506-556.qxd 6/5/10 9:10 AM Page 552

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554 Chapter 15. Enzymatic Catalysis

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Problems 555

1. Explain why ␥-pyridone is not nearly as effective a catalyst

for glucose mutarotation as is ␣-pyridone.What about ␤-pyridone?

2. RNA is rapidly hydrolyzed in alkaline solution to yield a

mixture of nucleotides whose phosphate groups are bonded to ei-

ther the 2¿ or the 3¿ positions of the ribose residues. DNA, which

lacks RNA’s 2¿ OH groups, is resistant to alkaline degradation.

Explain.

3. Carboxypeptidase A, a Zn

2⫹

-containing enzyme, hydrolyzes

the C-terminal peptide bonds of polypeptides (Fig 8-19a). In the

enzyme–substrate complex, the Zn

2⫹

ion is coordinated to three

enzyme side chains, the carbonyl oxygen of the scissile peptide

bond, and a water molecule. A plausible model for the enzyme’s

reaction mechanism that is consistent with X-ray and enzymolog-

ical data is diagrammed in Fig. 15-42. What are the roles of the

2⫹

ion and Glu 270 in this mechanism?

4. In the following lactonization reaction,

the relative reaction rate when R ⫽ CH

is 3.4 ⫻ 10

times that

when R ⫽ H. Explain.

*5. Derive the analog of Eq. [15.11] for an enzyme that cat-

alyzes the reaction:

Assume the enzyme must bind A before it can bind B:

6. Explain, in thermodynamic terms, why an “enzyme” that

stabilizes its Michaelis complex as much as its transition state does

not catalyze a reaction.

7. Suggest a transition state analog for proline racemase that

differs from those discussed in the text. Justify your suggestion.

8. Wolfenden has stated that it is meaningless to distinguish

between the “binding sites” and the “catalytic sites” of enzymes.

Explain.

9. Explain why oxalate (

⫺

OOCCOO

⫺

) is an inhibitor of ox-

aloacetate decarboxylase.

10. In light of the information given in this chapter, why are

enzymes such large molecules? Why are active sites almost always

E ⫹ A ⫹ B Δ EA ⫹ B Δ EAB S EP

A ⫹ B S P

CCH

COO

⫺

R R

PROBLEMS

Figure 15-42 Mechanism of carboxypeptidase A.

Glu 270 C

–

attack of

water

–

CHR

NH R⬘

Michaelis complex

Glu 270 C

–

H H

scissle bond

scission

–

CHR

NH R⬘

–

CHR

Glu 270 C

–

R⬘

Tetrahedral intermediate

Enzyme-product complex

Bioinformatics Exercises are available at www.wiley.com/college/voet.

Chapter 12

Enzyme Inhibitors and Rational Drug Design.

1. Dihydrofolate Reductase. Examine the structure of an enzyme with an inhibitor bound to it.

2. HIV Protease. Compare the structures of complexes containing HIV protease and an inhibitor.

3. Pharmacogenomics and Single Nucleotide Polymorphisms. Use online databases to find information on cytochrome P450 poly-

morphisms.

BIOINFORMATICS EXERCISES

JWCL281_c15_506-556.qxd 6/24/10 7:47 AM Page 555

located in clefts or depressions in enzymes rather than on protru-

sions?

11. Predict the effects on lysozyme catalysis of changing Phe

34, Ser 36, and Trp 108 to Arg, assuming that this change does not

significantly alter the structure of the protein.

*12. The incubation of (NAG)

with lysozyme results in the

slow formation of (NAG)

and (NAG)

. Propose a mechanism for

this reaction. What aspect of the lysozyme mechanism is sup-

ported by this reaction?

13. How would the lysozyme binding affinity of the following

(1 S 4)-linked tetrasaccharide compare with that of NAG–

NAM–NAG–NAM? Explain.

14. A major difficulty in investigating the properties of the

pancreatic serine proteases is that these enzymes, being proteins

themselves, are self-digesting. This problem is less severe, how-

ever, for solutions of chymotrypsin than it is for solutions of

trypsin or elastase. Explain.

15. The comparison of the active site geometries of chy-

motrypsin and subtilisin under the assumption that their similari-

ties have catalytic significance has led to greater mechanistic un-

derstanding of both these enzymes. Discuss the validity of this

strategy.

16. Benzamidine (K

 1.8  10

5

M) and leupeptin (K



1.8  10

7

M; Fig. 15-19)

Benzamidine

Leu

Leu NH CH CH

(CH

)

Leupeptin

H NHCOCH

NAGNAG NAM

are both specific competitive inhibitors of trypsin. Explain their

mechanisms of inhibition. Design leupeptin analogs that inhibit

chymotrypsin and elastase.

17. Trigonal boronic acid derivatives have a high tendency to

form tetrahedral adducts. 2-Phenylethyl boronic acid

is an inhibitor of subtilisin and chymotrypsin. Indicate the struc-

ture of these enzyme–inhibitor complexes.

18. Tofu (bean curd), a high-protein soybean product that is

widely consumed in China and Japan, is prepared in such a way as

to remove the trypsin inhibitor present in soybeans. Explain the

reason(s) for this treatment.

19. Explain why mutating all three residues of trypsin’s cat-

alytic triad has essentially no greater effect on the enzyme’s cat-

alytic rate enhancement than mutating only Ser 195.

20. Explain why chymotrypsin is not self-activating as is trypsin.

21. Does Lipinski’s “rule of five” predict that a hexapeptide

would be a therapeutically effective drug? Explain.

22. The preferred antidote for acetaminophen overdose is N-

acetylcysteine. Explain why the administration of this substance,

which must occur within 8 to 16 hours of the overdose, is an effec-

tive treatment.

23. Why would the activation of HIV-1 protease before the

virus buds from its host cell be disadvantageous to the virus?

Explain.

2–Phenylethyl boronic acid

556 Chapter 15. Enzymatic Catalysis

JWCL281_c15_506-556.qxd 6/5/10 9:10 AM Page 556

PART IV

METABOLISM

Schematic diagram of the major

pathways of energy metabolism.

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559

CHAPTER 16

Introduction to

Metabolism

1 Metabolic Pathways

2 Organic Reaction Mechanisms

A. Chemical Logic

B. Group-Transfer Reactions

C. Oxidations and Reductions

D. Eliminations, Isomerizations, and Rearrangements

E. Reactions That Make and Break Carbon–Carbon Bonds

3 Experimental Approaches to the Study of Metabolism

A. Metabolic Inhibitors, Growth Studies,

and Biochemical Genetics

B. Isotopes in Biochemistry

C. Isolated Organs, Cells, and Subcellular Organelles

D. Systems Biology

4 Thermodynamics of Phosphate Compounds

A. Phosphoryl-Transfer Reactions

B. Rationalizing the “Energy” in “High-Energy” Compounds

C. The Role of ATP

5 Oxidation–Reduction Reactions

A. The Nernst Equation

B. Measurements of Redox Potentials

C. Concentration Cells

6 Thermodynamics of Life

A. Living Systems Cannot Be at Equilibrium

B. Nonequilibrium Thermodynamics and the Steady State

C. Thermodynamics of Metabolic Control

Living organisms are not at equilibrium. Rather,they require

a continuous influx of free energy to maintain order in a uni-

verse bent on maximizing disorder. Metabolism is the over-

all process through which living systems acquire and utilize

the free energy they need to carry out their various func-

tions. They do so by coupling the exergonic reactions of nutri-

ent oxidation to the endergonic processes required to main-

tain the living state such as the performance of mechanical

work, the active transport of molecules against concentra-

tion gradients, and the biosynthesis of complex molecules.

How do living things acquire this necessary free energy?

And what is the nature of the energy coupling process?

Phototrophs (plants and certain bacteria; Section 1-1A)

acquire free energy from the sun through photosynthesis, a

process in which light energy powers the endergonic reac-

tion of CO

and H

O to form carbohydrates and O

(Chapter 24). Chemotrophs obtain their free energy by ox-

idizing organic compounds (carbohydrates, lipids, proteins)

obtained from other organisms, ultimately phototrophs.

This free energy is most often coupled to endergonic reac-

tions through the intermediate synthesis of “high-energy”

phosphate compounds such as adenosine triphosphate

(ATP; Section 16-4). In addition to being completely oxi-

dized, nutrients are broken down in a series of metabolic

reactions to common intermediates that are used as precur-

sors in the synthesis of other biological molecules.

A remarkable property of living systems is that, despite

the complexity of their internal processes, they maintain a

steady state. This is strikingly demonstrated by the observa-

tion that, over a 40-year time span, a normal human adult

consumes literally tons of nutrients and imbibes over 20,000 L

of water, but does so without significant weight change. This

steady state is maintained by a sophisticated set of metabolic

regulatory systems. In this introductory chapter to metabo-

lism, we outline the general characteristics of metabolic

pathways, study the main types of chemical reactions that

comprise these pathways, and consider the experimental

techniques that have been most useful in their elucidation.

We then discuss the free energy changes associated with re-

actions of phosphate compounds and oxidation–reduction

reactions. Finally we consider the thermodynamic nature

of biological processes, that is, what properties of life are

responsible for its self-sustaining character.

1 METABOLIC PATHWAYS

Metabolic pathways are series of consecutive enzymatic re-

actions that produce specific products. Their reactants, inter-

mediates, and products are referred to as metabolites. Since

an organism utilizes many metabolites, it has many meta-

bolic pathways. Figure 16-1 shows a metabolic map for a

typical cell with many of its interconnected pathways. Each

reaction on the map is catalyzed by a distinct enzyme, of

which there are ⬃4000 known.At first glance, this network

seems hopelessly complex. Yet, by focusing on its major

areas in the following chapters, for example, the main

pathways of glucose oxidation (the shaded areas of Fig.

16-1), we shall become familiar with its most important av-

enues and their interrelationships. Maps of metabolic path-

ways in a more readable form can be found on the Web

at http://www.expasy.org/cgi-bin/search-biochem-index,

http://www.iubmb-nicholson.org/, and http://www.genome.

ad.jp/kegg/metabolism.html.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 559

560 Chapter 16. Introduction to Metabolism

Figure 16-1 Map of the major metabolic pathways in a typical cell. The main pathways of

glucose metabolism are shaded. [Designed by Donald Nicholson. Published by BDH Ltd., Poole

2, Dorset, England.]

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