91

CHAPTER

7 Mechanisms and Inhibitors

7.1 A Few Basic Catalytic StrategiesAre Used by Many Enzymes

7.2 Enzyme Activity Can BeModulated by Temperature, pH,and Inhibitory Molecules

7.3 Chymotrypsin Illustrates BasicPrinciples of Catalysis andInhibition

Thus far, in our study of enzymes, we have learned that an enzyme binds asubstrate at the active site to facilitate the formation of the transition state; we

have developed a kinetic model for simple enzymes; and we learned that allostericenzymes are not only catalysts, but also information sensors. We now turn ourattention to the catalytic strategies of enzymes and then to an examination of howenzyme activity can be modulated by environmental factors distinct fromallosteric signals. Finally, we consider the catalytic mechanism of the digestiveenzyme chymotrypsin, one of the first enzymes understood in mechanistic detail.

7.1 A Few Basic Catalytic Strategies Are Used by Many Enzymes

As we will see in our study of biochemistry, enzymes catalyze a vast array ofchemical reactions. Despite this diversity, all enzymes operate by facilitating theformation of the transition state. How the transition state is formed varies fromenzyme to enzyme, but most enzymes commonly employ one or more of thefollowing strategies to catalyze specific reactions:

1. Covalent Catalysis. In covalent catalysis, the active site contains a reactivegroup, usually a powerful nucleophile that becomes temporarily covalently

Chess and enzymes have in common the use of strategy, consciously thought out in the gameof chess and selected by evolution for the action of an enzyme. In the chess depicted here, theking is trapped as a result of a number of strategic moves. Likewise, enzymes have catalyticstrategies, developed over evolutionary time, for binding their substrates and chemically actingon them. [Vladimir Mucibabic/Feature Pics.]

A nucleophile is a chemical that isattracted to regions of positive charge inanother molecule. A nucleophileparticipates in a chemical reaction bydonating electrons to another chemical,called the electrophile.

modified in the course of catalysis. The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism (p. 100).

2. General Acid–Base Catalysis. In general acid–base catalysis, a molecule other thanwater plays the role of a proton donor or acceptor. Chymotrypsin uses a histidineresidue as a base catalyst to enhance the nucleophilic power of serine (p. 102).

3. Metal Ion Catalysis. Metal ions can function catalytically in several ways. Forinstance, a metal ion may serve as an electrophilic catalyst, stabilizing a negativecharge on a reaction intermediate. Alternatively, the metal ion may generate anucleophile by increasing the acidity of a nearby molecule, such as water. Finally,the metal ion may bind to the substrate, increasing the number of interactionswith the enzyme and thus the binding energy. Metal ions are required cofactorsfor many of the enzymes that we will encounter in our study of biochemistry.

4. Catalysis by Approximation. Many reactions include two distinct substrates. Insuch cases, the reaction rate may be considerably enhanced by bringing the twosubstrates into proximity on a single binding surface of an enzyme.

Recall from Chapter 5 that we considered another key aspect of enzyme catalysis—binding energy. The full complement of binding interactions betweenan enzyme and a substrate is formed only when the substrate is in the transitionstate. The fact that binding energy is maximal when the enzyme binds to the transi-tion state favors the formation of the transition state and thereby promotes catalysis.

7.2 Enzyme Activity Can Be Modulated by Temperature, pH,and Inhibitory Molecules

Regardless of which mechanism or mechanisms employed by an enzyme to catalyzea reaction, the rate of catalysis is affected by the same environmental parametersthat affect all chemical reactions, such as temperature and pH. Moreover, somechemicals that interact specifically with the elaborate three-dimensional structureof the enzyme also can affect enzyme activity.

Temperature Enhances the Rate of Enzyme-Catalyzed ReactionsAs the temperature rises, the rate of most reactions increases. The rise in temper-ature increases the Brownian motion of the molecules, which makes interactionsbetween an enzyme and its substrate more likely. For most enzymes, there is atemperature at which the increase in catalytic activity ceases and there is precip-itous loss of activity (Figure 7.1). What is the basis of this loss of activity? Recallthat proteins have a complex three-dimensional structure that is held togetherby weak bonds. When the temperature is increased beyond a certain point, theweak bonds maintaining the three-dimensional structure are not strong enough

Enzy

me

activ

ity

Temperature

Figure 7.1 The effect of heat on enzymeactivity. The enzyme tyrosinase, which ispart of the pathway that synthesizes thepigment that results in dark fur, has a lowtolerance for heat. It is inactive at normalbody temperatures but functional atslightly lower temperatures. Theextremities of a Siamese cat are coolenough for tyrosinase to gain function andproduce pigment. [Photograph: JaneBurton/Getty.]

An electrophile is a chemical that iselectron deficient. Electrophiles are thusattracted to nucleophiles—electron-richmolecules or regions of molecules.

927 Mechanisms and Inhibitors

Figure 7.3 Thermophilic archaea and theirenvironment. Archaea can thrive inhabitats as harsh as a volcanic vent. Here,the archaea form an orange matsurrounded by yellow sulfurous deposits.[Krafft-Explorer/Photo Researchers.]

to withstand the polypeptide chain’s thermal jostling and the protein loses thestructure required for activity. The protein is said to be denatured (p. 55).

In organisms such as ourselves that maintain a constant body temperature(endotherms), the effect of outside temperature on enzyme activity is minimized.However, in organisms that assume the temperature of the ambient environment(ectotherms), temperature is an important regulator of biochemical and, indeed,biological activity. Lizards, for instance, are most active in warmer temperaturesand relatively inactive in cooler temperatures, a behavioral manifestation of bio-chemical activity (Figure 7.2).

Some organisms, such as thermophilic archaea, can live at temperatures of80°C, temperatures that would denature most proteins. The proteins in theseorganisms have evolved to be very resistant to thermal denaturation (Figure 7.3).

Most Enzymes Have an Optimal pHEnzyme activity also often varies with pH, the H� concentration of the environment (p. 24). The activity of most enzymes displays a bell-shaped curvewhen examined as a function of pH. However, the optimal pH—the pH at whichenzymes display maximal activity—varies with the enzyme and is correlated withthe environment of the enzyme. For instance, the protein-digesting enzymepepsin functions in the very acidic environment of the stomach, where the pH isbetween 1 and 2. Most enzymes would be denatured at this pH, but pepsin func-tions very effectively. The enzymes of the pancreatic secretion of the upper smallintestine, such as chymotrypsin, have pH optima near pH 8, in keeping with thepH of the intestine in this region (Figure 7.4).

How can we account for the pH effect on enzyme activity in regard to ourunderstanding of enzymes in particular and proteins in general? Imagine anenzyme that requires ionization of both a glutamic acid residue and an arginineresidue at the active site for the enzyme to be functional. Thus, the enzyme woulddepend on the presence of a group as well as an group (Figure 7.5). If the pH is lowered (the H� concentration increases), the groups will gradually be converted into groups with a concomitant lossof enzyme activity. On the other side of the optimum, as the pH is raised (less H�,more OH�, the group loses an H� to OH�, becoming a neutral group, and the enzyme activity is diminished. Often, the activity-versus-pH curvesare due to several ionizable groups.

¬NH2¬NH3+

¬COOH

¬COO-¬NH3

+¬COO-

Figure 7.2 A lizard basking in the sun.Ectotherms, such as the Namibian rockagama (Agama planiceps), adjust theirbody temperatures and, hence, the rate ofbiochemical reactions behaviorally.[Morales/Age Fotostock.]

Figure 7.4 The pH dependence of the activity of the enzymes pepsin andchymotrypsin. Chymotrypsin and pepsin have different optimal pH values. Theoptimal pH for pepsin is noteworthy. Most proteins would be denatured at thisacidic pH.

Enzy

me

activ

ity

111 2 3 4 5 6 7 8 9 100

pH

Pepsin Chymotrypsin

93

NH

3 +N

H2 + H + CO

OH

COO

−+

H+

Enzy

me

activ

ity

142 4 6 8 10 120

pH

Figure 7.5 A pH profile for a hypotheticalenzyme. The pH dependence of enzymes is due to the presence of ionizable Rgroups.

Figure 7.6 Reversible inhibitors. (A) Substrate binds to an enzyme’s active site toform an enzyme–substrate complex. (B) A competitive inhibitor binds at the activesite and thus prevents the substrate from binding. (C) An uncompetitive inhibitorbinds only to the enzyme–substrate complex. (D) A noncompetitive inhibitor doesnot prevent the substrate from binding.

Is alteration of the pH of the cellular milieu ever used as a regulatordevice? The answer is yes, and a crucial enzyme in glucose metabolism inskeletal muscle provides an example. Phosphofructokinase (Chapter 15)controls the rate of metabolism of glucose under aerobic conditions (in thepresence of oxygen) as well as anaerobic conditions (in the absence of oxy-gen). A problem arises with the rapid processing of glucose in the absenceof oxygen: the end product is lactic acid, which readily ionizes to lactate anda hydrogen ion. To prevent the muscle from becoming “pickled”by the highconcentration of acid, the activity of the phosphofructokinase decreases ifthe pH falls too drastically, which, in turn, reduces the metabolism of glu-cose to lactic acid. Phosphofructokinase is made up of multiple subunits,and the decrease in pH causes the subunits to dissociate, rendering theenzyme inactive and thus reducing lactic acid production.

Enzymes Can Be Inhibited by Specific MoleculesThe binding of specific small molecules and ions can inhibit the activity ofmany enzymes. The regulation of allosteric enzymes typifies this type ofcontrol (p. 81). In regard to allosteric enzymes, the interaction of the signalmolecule and the enzyme is the result of the evolutionary process. In addi-tion to the evolutionarily derived signal molecules, many drugs and toxicagents act by inhibiting enzymes. Inhibition by particular chemicals can bea source of insight into the mechanism of enzyme action: specificinhibitors can often be used to identify residues critical for catalysis.

Enzyme inhibition can be either reversible or irreversible. We beginthe investigation of enzyme inhibition by first examining reversible inhi-bition. In contrast with irreversible inhibition, reversible inhibition is char-acterized by a rapid dissociation of the enzyme–inhibitor complex. Thereare three common types of reversible inhibition: competitive inhibition,uncompetitive inhibition, and noncompetitive inhibition. These threetypes of inhibition differ in the nature of the interaction between theenzyme and the inhibitor and in the effect that the inhibitor has on enzymekinetics (Figure 7.6). We will consider each of these types in turn.

In competitive inhibition, the inhibitor resembles the substrate and binds tothe active site of the enzyme (see Figure 7.6B). The substrate is thereby preventedfrom binding to the same active site. An enzyme can bind substrate (forming anES complex) or inhibitor (EI), but not both (ESI). A competitive inhibitor dimin-ishes the rate of catalysis by reducing the proportion of enzyme molecules bound to asubstrate. At any given inhibitor concentration, competitive inhibition can berelieved by increasing the substrate concentration. Under these conditions, thesubstrate “out competes” the inhibitor for the active site.

Some competitive inhibitors are useful drugs. One of the earliest exampleswas the use of sulfanilamide as an antibiotic. Sulfanilamide is an example of a sulfadrug, a sulfur-containing antibiotic. Structurally, sulfanilamide mimicsp-aminobenzoic acid (PABA), a metabolite required by bacteria for the synthesisof folic acid. Sulfanilamide binds to the enzyme that normally metabolizes PABAand competitively inhibits it, preventing folic acid synthesis. Human beings,

Substrate

Substrate

Enzyme

Substrate

Enzyme

Enzyme

Enzyme

Noncompetitiveinhibitor

Uncompetitiveinhibitor

Competitiveinhibitor

(A)

(B)

(C)

(D)

NH2

NH2

OHO

O S

NH2

CO

Sulfanilamide PABA

94

unlike bacteria, absorb folic acid from the diet and are thus unaffected by the sulfadrug. Other competitive inhibitors commonly used as drugs include ibuprofen,which inhibits a cyclooxygenase that helps to generate the inflammatory response,and statins, which inhibit the key enzyme in cholesterol synthesis.

Uncompetitive inhibition is distinguished by the fact that the inhibitor bindsonly to the enzyme–substrate complex. The uncompetitive inhibitor’s bindingsite is created only when the enzyme binds the substrate (see Figure 7.6C).Uncompetitive inhibition cannot be overcome by the addition of more substrate.The herbicide glyphosate, also known as Roundup, is an uncompetitive inhibitorof an enzyme in the biosynthetic pathway for aromatic amino acids in plants. Theplant dies because it lacks these amino acids.

In noncompetitive inhibition, the inhibitor and substrate can bind simultane-ously to an enzyme molecule at different binding sites (see Figure 7.6D). A non-competitive inhibitor acts by decreasing the overall number of active enzymemolecules rather than by diminishing the proportion of enzyme molecules that arebound to substrate. Noncompetitive inhibition, in contrast with competitive inhi-bition, cannot be overcome by increasing the substrate concentration. Deoxycy-cline, an antibiotic, functions at low concentrations as a noncompetitive inhibitorof a bacterial proteolytic enzyme (collagenase). Inhibition of this enzyme preventsthe growth and reproduction of bacteria that cause gum (periodontal) disease.

Reversible Inhibitors Are Kinetically DistinguishableHow can we determine whether a reversible inhibitor acts by competitive,uncompetitive, or noncompetitive inhibition? Let us consider only enzymesthat exhibit Michaelis–Menten kinetics—that is, enzymes that are not alloster-ically inhibited. Measurements of the rates of catalysis at different concentra-tions of substrate and inhibitor serve to distinguish the three types of reversibleinhibition. In competitive inhibition, the inhibitor competes with the substratefor the active site. The hallmark of competitive inhibition is that it can be over-come by a sufficiently high concentration of substrate (Figure 7.7). The effect ofa competitive inhibitor is to increase the apparent value of KM, meaning thatmore substrate is needed to obtain the same reaction rate. This new apparentvalue of KM is called KM

app. In the presence of a competitive inhibitor, an enzymewill have the same Vmax as in the absence of an inhibitor. At a sufficiently highconcentration, virtually all the active sites are filled by substrate, and theenzyme is fully operative. The more inhibitor is present, the more substrate isrequired to displace it and reach Vmax.

In uncompetitive inhibition, the inhibitor binds only to the ES complex. Thisenzyme–substrate–inhibitor complex, ESI, does not proceed to form any prod-uct. Because some unproductive ESI complex will always be present, Vmax willbe lower in the presence of inhibitor than in its absence (Figure 7.8). Theuncompetitive inhibitor also lowers the apparent value of KM, because theinhibitor binds to ES to form ESI, depleting ES. To maintain the equilibriumbetween E and ES, more S binds to E. Thus, a lower concentration of S isrequired to form half of the maximal concentration of ES, resulting in a reduc-tion of the apparent value of KM, now called KM

app. Likewise, the value of Vmaxis decreased to a new value called Vmax

app .

ES

Rela

tive

rate

S

No inhibitor

+10[I]

+[ I]

+5[I]

EI

I

S

[Substrate]

100

80

60

40

20

0

E + PE+I

Ki

Figure 7.7 Kinetics of a competitiveinhibitor. As the concentration of acompetitive inhibitor increases, higherconcentrations of substrate are required toattain a particular reaction velocity. Thereaction pathway suggests how sufficientlyhigh concentrations of substrate cancompletely relieve competitive inhibition.

ES + I

Rela

tive

rate

No inhibitor

+10[I]

+[ I]

+5[I]

S

ESI

[Substrate]

100

80

60

40

20

0

E + PE + I

Ki

KM for uninhibited enzyme

KMapp for [ I] = Ki

Figure 7.8 Kinetics of an uncompetitive inhibitor. The reaction pathway shows that theinhibitor binds only to the enzyme–substrate complex. Consequently, Vmax cannot beattained, even at high substrate concentrations. The apparent value for KM is lowered,becoming smaller as more inhibitor is added.

957.2 Enzyme Inhibition

ES

Rela

tive

rate

No inhibitor

+10[I]

+[ I]

+5[I]

EI

S

ESIS

[Substrate]

100

80

60

40

20

0

E + PE + I

Ki

Figure 7.9 Kinetics of a noncompetitiveinhibitor. The reaction pathway shows thatthe inhibitor binds both to free enzymeand to enzyme complex. Consequently, aswith uncompetitive competition, Vmaxcannot be attained. KM remainsunchanged, and so the reaction rateincreases more slowly at low substrateconcentrations than is the case foruncompetitive competition.

1/V

1/[S]0

+ Competitiveinhibitor

No inhibitorpresent

Figure 7.10 Competitive inhibitionillustrated on a double-reciprocal plot. Adouble-reciprocal plot of enzyme kinetics inthe presence and absence of a competitiveinhibitor illustrates that the inhibitor hasno effect on Vmax but increases KM.

1/V

1/[S ]0

+ Uncompetitiveinhibitor

No inhibitorpresent

Figure 7.11 Uncompetitive inhibitionillustrated on a double-reciprocal plot. Anuncompetitive inhibitor does not affectthe slope of the double-reciprocal plot.Vmax and KM are reduced by equivalentamounts.

1/V

1/[S]0

+ Noncompetitiveinhibitor

No inhibitorpresent

Figure 7.12 Noncompetitive inhibitionillustrated on a double-reciprocal plot. Adouble-reciprocal plot of enzyme kineticsin the presence and absence of anoncompetitive inhibitor shows that KM isunaltered and Vmax is decreased.

967 Mechanisms and Inhibitors

In noncompetitive inhibition (Figure 7.9), a substrate can bind to theenzyme–inhibitor complex as well as to the enzyme alone. In either case, theenzyme–inhibitor–substrate complex does not proceed to form product.The value of Vmax is decreased to the new value whereas the value of KM isunchanged. Why is Vmax lowered though KM remains unchanged? In essence, theinhibitor simply lowers the concentration of functional enzyme. The resultingsolution behaves like a more-dilute solution of enzyme. Noncompetitive inhibitioncannot be overcome by increasing the substrate concentration.

Double-reciprocal plots are especially useful for distinguishing competitive,uncompetitive, and noncompetitive inhibitors. In competitive inhibition, the inter-cept on the y-axis, 1/Vmax, is the same in the presence and in the absence of inhibitor,although the slope (KM/Vmax) is increased (Figure 7.10). The intercept is unchangedbecause a competitive inhibitor does not alter Vmax. The increase in the slope of the1/V0 versus 1/[S] plot indicates the strength of binding of competitive inhibitor.

In uncompetitive inhibition (Figure 7.11), the inhibitor combines only with theenzyme–substrate complex. The slope of the line is the same as that for the unin-hibited enzyme, but the intercept on the y-axis, 1/Vmax, is increased. Consequently,for uncompetitive inhibition, the lines in double-reciprocal plots are parallel.

In noncompetitive inhibition (Figure 7.12), the inhibitor can combine witheither the enzyme or the enzyme–substrate complex. In pure noncompetitive

Vappmax ,

inhibition, the values of the dissociation constants of the inhibitor and enzymeand of the inhibitor and enzyme–substrate complex are equal. The value of Vmaxis decreased to the new value and so the intercept on the vertical axis isincreased. The slope when the inhibitor is present, which is equal to islarger by the same factor. In contrast with Vmax, KM is not affected by pure non-competitive inhibition.

Irreversible Inhibitors Can Be Used to Map the Active SiteWhereas a reversible inhibitor will both bind to an enzyme and dissociate from itrapidly, an irreversible inhibitor dissociates very slowly from its target enzymebecause it has become tightly bound to the enzyme, either covalently or nonco-valently. Some irreversible inhibitors are important drugs. Penicillin acts by cova-lently modifying the enzyme transpeptidase, thereby preventing the synthesis ofbacterial cell walls and thus killing the bacteria (p. 98). Aspirin acts by covalentlymodifying the enzyme cyclooxygenase (the same enzyme inhibited by ibuprofen),reducing the synthesis of inflammatory signals.

Irreversible inhibitors that covalently bind to an enzyme provide a means forelucidating the mechanism of enzymes. The first step in determining the chemi-cal mechanism of an enzyme is to determine which functional groups are requiredfor enzyme activity. Irreversible inhibitors modify functional groups, which canthen be identified. If treatment with an irreversible inhibitor results in a loss ofenzyme activity, then this loss suggests that the modified group is required forenzyme activity. Irreversible inhibitors can be assorted into four categories: group-specific reagents, affinity labels (substrate analogs), suicide inhibitors, and transi-tion-state analogs.

Group-specific reagents react with specific R groups of amino acids. An exampleof a group-specific reagent is diisopropylphosphofluoridate (DIPF). DIPF inhibitsthe proteolytic enzyme chymotrypsin by modifying only 1 of the 28 serine residuesin the protein, implying that this serine residue is especially reactive. As we will seeon page 101, it is indeed the case that this serine residue is at the active site. DIPF alsorevealed a reactive serine residue in acetylcholinesterase, an enzyme important in thetransmission of nerve impulses (Figure 7.13). Thus, DIPF and similar compoundsthat bind and inactivate acetylcholinesterase are potent nerve gases.

KM/Vappmax ,

Vappmax ,

QUICK QUIZ 1 Identify the curvein the graph below that corresponds

to each of the following conditions: noinhibition, competitive inhibition,noncompetitive inhibition, uncompetitiveinhibition.

+

DIPF

P

O

O

O PO

F

HC

H3

3

C

H

O

HC

H3

3

C

H

CH3

CH3

H

P

OO

CH3

CH3

H

OH

Acetylcholin-esterase

Inactivatedenzyme

+ F– + H+Ser

Figure 7.13 Enzyme inhibition bydiisopropylphosphofluoridate (DIPF), agroup-specific reagent. DIPF can inhibit anenzyme by covalently modifying a crucialserine residue.

Effects of inhibitors on a plot of V0 versus [S]

1

2

3

4

[S]

V 0

Affinity labels, also called substrate analogs, are molecules that covalently mod-ify active-site residues and are structurally similar to the substrate for an enzyme.They are thus more specific for an enzyme’s active site than are group-specificreagents. Tosyl-L-phenylalanine chloromethyl ketone (TPCK) is an affinity label forchymotrypsin (Figure 7.14). TPCK binds at the active site and then reacts irre-versibly with a histidine residue at that site, inhibiting the enzyme.

97

Suicide inhibitors, or mechanism-based inhibitors, are chemically modifiedsubstrates. These molecules provide researchers with the most-specific means ofmodifying an enzyme’s active site. The inhibitor binds to the enzyme as a substrateand is initially processed by the normal catalytic mechanism. The mechanism ofcatalysis then generates a chemically reactive intermediate that inactivates theenzyme through covalent modification. The fact that the enzyme participates inits own irreversible inhibition strongly suggests that the covalently modifiedgroup on the enzyme is catalytically vital. The antibiotic penicillin is a suicideinhibitor of the enzyme that synthesizes bacterial cell walls.

Transition-state analogs are potent inhibitors of enzymes (Chapter 5). Asdiscussed earlier, the formation of the transition state is crucial to enzyme catal-ysis (p. 71). An important piece of evidence supporting the role of transition-stateformation in catalysis is the inhibitory power of transition-state analogs.

Clinical Insight

Penicillin Irreversibly Inactivates a Key Enzyme in Bacterial Cell-Wall SynthesisPenicillin, the first antibiotic discovered, consists of a thiazolidine ring fusedto a �-lactam ring to which a variable R group is attached by a peptide bond(Figure 7.15). This structure can undergo a variety of rearrangements, and, inparticular, the �-lactam ring is very unstable. Indeed, this instability is closelytied to the antibiotic action of penicillin, as will be evident shortly.

How does penicillin inhibit bacterial growth? Let us consider Staphylococcusaureus, the most common cause of staph infections. Penicillin works by interfer-ing with the synthesis of the S. aureus cell walls. The S. aureus cell wall is madeup of a macromolecule, called a peptidoglycan (Figure 7.16), which consists oflinear polysaccharide chains that are cross-linked by short peptides (pen-taglycines and tetrapeptides). The enormous bag-shaped peptidoglycan confersmechanical support and prevents bacteria from bursting in response to their highinternal osmotic pressure. Glycopeptide transpeptidase catalyzes the formation of

987 Mechanisms and Inhibitors

Reactive peptidebond in

ββ-lactam ring

Thiazolidinering

R

Variablegroup

N

S

O

HN

O

H

C

C

CH3

CH3

COO–

Figure 7.15 The structure of penicillin. Thereactive site of penicillin is the peptidebond of its �-lactam ring.

NH

H

O

S

H C

O O

NH

H

C

C

C

O

HNR�

R�

HN

N

N

N

O

R

Cl

3

Specificity group

Reactive group

Natural substrate for chymotrypsin

Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

Chymotrypsin

His 57

+TPCK

(A) (B)

Figure 7.14 Affinity labeling. (A) Tosyl-L-phenylalanine chloromethyl ketone (TPCK)is a reactive analog of the normalsubstrate for the enzyme chymotrypsin. (B) TPCK binds at the chymotrypsin activesite and modifies an essential histidineresidue.

the cross-links that make the peptidoglycan so stable (Figure 7.17). Bacterial cellwalls are unique in containing D amino acids, which form cross-links by a mech-anism different from that used to synthesize proteins.

Figure 7.17 The formation of cross-links in S. aureus peptidoglycan. The terminal aminogroup of the pentaglycine bridge in the cell wall attacks the peptide bond between twoD-alanine residues to form a cross-link.

Figure 7.16 A schematic representationof the peptidoglycan in Staphylococcusaureus. The sugars are shown in yellow,the tetrapeptides in red, and thepentaglycine bridges in blue. The cellwall is a single, enormous, bag-shapedmacromolecule because of extensivecross-linking.

RC C

CH2

O

RC

O

NH3+ R�

NH

C

O

C

O

HN

O

O

HCH3

CH3H

R�NH

HN

CH2

HCH3

–NH2

CH3H

CO

O

–

+

Terminal glycineresidue of

pentaglycinebridge

Terminal D-Ala-D-Ala unit

+

Gly-D-Ala cross-link D-Ala

NH

CC

O

C

OHN

R�

O

O

CH3H

C

OHN

R�

CH3H

NH

H2C

C

HN

R�

O

R

CH3H

H3CH

–

D-Ala D-Ala

D-AlaEnzyme

enzyme

Acyl-enzyme intermediateTerminal D-Ala-D-Ala Gly-D-Ala cross-link

Terminal glycine

RC

O

H2C

H2N

EnzymeGly

Figure 7.18 A transpeptidation reaction. An acyl-enzyme intermediate is formed in thetranspeptidation reaction leading to cross-link formation.

997.2 Enzyme Inhibition

Penicillin inhibits the cross-linking transpeptidase by the Trojan horse strat-agem: it mimics a normal substrate to enter the active site. To create cross-linksbetween the tetrapeptides and pentaglycines, the transpeptidase normally formsan acyl intermediate with the penultimate D-alanine residue of the tetrapeptide.This covalent acyl-enzyme intermediate then reacts with the amino group of theterminal glycine in another peptide to form the cross-link (Figure 7.18).Penicillin is welcomed into the active site of the transpeptidase because it mim-ics the D-Ala-D-Ala moiety of the normal substrate. On binding to the transpep-tidase, the serine residue at the active site attacks the carbonyl carbon atomof the lactam ring to form the penicilloyl-serine derivative (Figure 7.19).

This penicilloyl-enzyme does not react further. Hence, the transpeptidase is irre-versibly inhibited and cell-wall synthesis cannot take place. Because the pepti-dase participates in its own inactivation, penicillin acts as a suicide inhibitor. ■

7.3 Chymotrypsin Illustrates Basic Principles of Catalysis and Inhibition

A detailed examination of the mechanism of action of the protein-degradingenzyme chymotrypsin will illustrate some of the basic principles of catalysis. Itwill also be useful as a case study for showing how enzyme mechanisms can beinvestigated, including the use of kinetics and enzyme inhibitors.

Protein turnover is an important process in living systems. After they are nolonger needed in the cell, proteins must be degraded so that their constituentamino acids can be recycled for the synthesis of new proteins. Additionally,proteins ingested in the diet must be broken down into small peptides and aminoacids for absorption in the intestine. Protein breakdown is catalyzed by a large classof enzymes called proteolytic enzymes or proteases. These enzymes cleave proteinsby a hydrolysis reaction—the addition of a molecule of water to a peptide bond.

One such enzyme is chymotrypsin, which is secreted by the pancreas inresponse to a meal. Chymotrypsin cleaves peptide bonds selectively on the carboxyl-terminal side of the large hydrophobic amino acids such as tryptophan,tyrosine, phenylalanine, and methionine (Figure 7.20).

1007 Mechanisms and Inhibitors

+H3N C

HN

NH

HN

HN

NH

CC

C

CC

O

O

O

O

O

C

O

O

H3CH

H

H2CH

CH2

H

H

H2C

H

CH2

S

CH3

NH2

O

HO

CH2

H2C

C O

O–

–

Ala Phe Asn Ser Met Glu

CH2Figure 7.20 The specificity ofchymotrypsin. Chymotrypsin cleavesproteins on the carboxyl side of aromaticor large hydrophobic amino acids (shadedorange). The red bonds indicate wherechymotrypsin most likely acts.

Figure 7.19 The formation of a penicilloyl-enzyme complex. Penicillin reacts with thetranspeptidase to form an inactivecomplex, which is indefinitely stable.

NH

COO–

CH3

CH3

C

O

O

NHH

CO

R

OHSer

Glycopeptidetranspeptidase

Penicilloyl-enzyme complex(enzymatically inactive)

Penicillin

Serine 195 Is Required for Chymotrypsin ActivityChymotrypsin is a good example of the use of covalent modification as a catalyticstrategy. The enzyme employs a powerful nucleophile to attack the unreactivecarbonyl group of the substrate. This nucleophile becomes covalently attached tothe substrate briefly in the course of catalysis.

101

+ –O N

O

O

NH

CC

O

H3C

O

O

N

O

O

H2CH

NH

CC

O

H3C

O

H2CH

OH+ H +

+H2O

N-Acetyl-L-phenylalanine p-nitrophenyl ester p-Nitrophenolate

Figure 7.21 A chromogenic substrate. N-Acetyl-L-phenylalanine p-nitrophenyl ester yields ayellow product, p-nitrophenolate, on cleavage by chymotrypsin. p-Nitrophenolate forms bydeprotonation of p-nitrophenol at pH 7.

Steady-state phase

Milliseconds after mixing

Burst phase

Abso

rban

ce(p

-nitr

ophe

nol r

elea

sed)

Figure 7.22 Kinetics of chymotrypsincatalysis. Two stages are evident in thecleaving of N-acetyl-L-phenylalanine p-nitrophenyl ester by chymotrypsin: arapid burst phase (pre-steady state) anda steady-state phase.

+

XH

OH

Enzyme

Acylation

Acyl-enzyme

XH = ROH (ester),RNH2 (amide)

Deacylation

H2O

X

R

O

O

R

O

HO

R

O

+OH

Enzyme

(A) (B)

C C C Figure 7.23 Covalent catalysis. Hydrolysisby chymotrypsin takes place in two stages:(A) acylation to form the acyl-enzymeintermediate followed by (B) deacylationto regenerate the free enzyme.

What is the nucleophile that chymotrypsin employs to attack the substratecarbonyl group? A clue came from the fact that chymotrypsin contains an extra-ordinarily reactive serine residue. Treatment with organofluorophosphates thatmodify serine residues, such as DIPF (p. 97), was found to inactivate the enzymeirreversibly (see Figure 7.13). Despite the fact that the enzyme possesses 28 serineresidues, only one of them, serine 195, was modified, resulting in a total loss ofenzyme activity. The use of the group-specific reagent DIPF alerted researchers tothe importance of one particular serine residue in catalysis.

Chymotrypsin Action Proceeds in Two Steps Linked by a Covalently Bound IntermediateA study of the enzyme’s kinetics suggested a role for serine 195. The kinetics ofenzyme action are often easily monitored by having the enzyme act on a substrateanalog that forms a colored product. For chymotrypsin, such a chromogenicsubstrate is N-acetyl-L-phenylalanine p-nitrophenyl ester. One of the productsformed by chymotrypsin’s cleavage of this substrate is p-nitrophenolate, whichhas a yellow color (Figure 7.21). Measurements of the absorbance of light revealedthe amount of p-nitrophenolate being produced and thus provided a facile meansof investigating chymotrypsin activity.

Under steady-state conditions, the cleavage of the substrate obeysMichaelis–Menten kinetics. More-insightful results were obtained by examiningproduct formation within milliseconds of mixing the enzyme and substrate. Thereaction between chymotrypsin and N-acetyl-L-phenylalanine p-nitrophenyl esterproduced an initial rapid burst of colored product, followed by its slower forma-tion as the reaction reached the steady state (Figure 7.22). These results suggestthat hydrolysis proceeds in two steps. The burst is observed because the first stepis more rapid than the second step.

The two steps are explained by the formation of a covalently boundenzyme–substrate intermediate (Figure 7.23). First, the acyl group of the substratebecomes covalently attached to serine 195 of the enzyme as p-nitrophenolate isreleased. The enzyme–acyl-group complex is called the acyl-enzyme intermediate.

In a steady-state system, theconcentrations of the intermediatesstay the same, even though theconcentrations of substrate andproducts are changing. A sink filledwith water that has the tap open justenough to match the loss of waterdown the drain is in a steady state.

Second, the acyl-enzyme intermediate is hydrolyzed to release the carboxylic acidcomponent of the substrate and regenerate the free enzyme. Thus, one moleculeof p-nitrophenolate is produced rapidly from each enzyme molecule as the acyl-enzyme intermediate is formed. However, it takes longer for the enzyme to be“reset” by the hydrolysis of the acyl-enzyme intermediate, and both steps arerequired for enzyme turnover. Note that chymotrypsin catalysis proceeds througha substituted-enzyme intermediate, the hallmark of a double-displacement, orping-pong, reaction mechanism.

The Catalytic Role of Histidine 57 Was Demonstrated by Affinity LabelingThe importance of a second residue in catalysis was shown by affinity labeling.The strategy was to have chymotrypsin react with a molecule that (1) specificallybinds to the active site because it resembles a substrate and then (2) forms a sta-ble covalent bond with a group on the enzyme that is in proximity. These criteriaare met by TPCK (see Figure 7.14). The phenylalanine side chain of TPCK enablesit to bind specifically to chymotrypsin. The reactive group in TPCK is the chloro-methyl ketone, which covalently modifies one of the ring nitrogens of histidine57. TPCK is positioned to react with this residue because of its specific binding tothe active site of the enzyme. The TPCK derivative of chymotrypsin is enzymati-cally inactive.

Serine Is Part of a Catalytic Triad That Includes Histidine and Aspartic AcidThus far, we have learned that serine 195 and histidine 57 are required for chymotrypsin activity, and the reaction proceeds through a substituted-enzymeintermediate. How can we integrate this information to elucidate the mecha-nism of chymotrypsin action? The side chain of serine 195 is hydrogen bondedto the imidazole ring of histidine 57. The group of this imidazole ring is,in turn, hydrogen bonded to the carboxylate group of aspartate 102, another keycomponent of the active site. This constellation of residues is referred to as thecatalytic triad.

How does this arrangement of residues lead to the high reactivity of serine195? The histidine residue serves to position the serine side chain and to polarizeits hydroxyl group so that it is poised for deprotonation. In the presence of thesubstrate, histidine 57 accepts the proton from the serine-195 hydroxyl group. Indoing so, histidine acts as a general base catalyst. The withdrawal of the protonfrom the hydroxyl group generates an alkoxide ion, which is a much more pow-erful nucleophile than a hydroxyl group is. The aspartate residue helps orient thehistidine residue and make it a better proton acceptor through hydrogen bondingand electrostatic effects (Figure 7.24).

¬NH

1027 Mechanisms and Inhibitors

Asp 102 His 57 Ser 195

OH

NN

O

CO H

–

–ONN

O

CO H H

– +

Alkoxideion

Figure 7.24 The catalytic triad. The catalytic triad, shown on the left, converts serine 195into a potent nucleophile, as illustrated on the right.

These observations suggest a mechanism for peptide hydrolysis (Figure 7.25).After substrate binding (step 1), the reaction begins with the oxygen atom of theside chain of serine 195 making a nucleophilic attack on the carbonyl carbon atomof the target peptide bond (step 2). There are now four atoms bonded to the car-bonyl carbon atom, arranged as a tetrahedron, instead of three atoms in a planararrangement. The inherently unstable tetrahedral-intermediate form bears a neg-ative charge on the oxygen atom derived from the carbonyl group. This charge isstabilized by interactions with NH groups from the protein in a site termed theoxyanion hole (Figure 7.26). These interactions contribute to the binding energythat helps stabilize the transition state that precedes the formation of the tetrahe-dral intermediate. This tetrahedral intermediate collapses to generate theacyl-enzyme (step 3). This step is facilitated by the transfer of the proton from thepositively charged histidine residue, now acting as a general acid catalyst, to theamino group of the substrate formed by cleavage of the peptide bond. The aminecomponent is now free to depart from the enzyme (step 4), completing the firststage of the hydrolytic reaction—that is, acylation of the enzyme.

The next stage—deacylation of the enzyme—begins when a water moleculetakes the place occupied earlier by the amine component of the substrate (step 5).The ester group of the acyl-enzyme is now hydrolyzed by a process that essentially

Oxyanion hole

Gly 193

Ser 195−

Figure 7.26 The oxyanion hole. Thestructure stabilizes the tetrahedralintermediate of the chymotrypsin reaction.Notice that hydrogen bonds (shown ingreen) link peptide NH groups and thenegatively charged oxygen atom of theintermediate.

103

Oxyanionhole

Oxyanionhole

Acyl-enzyme

2

Tetrahedralintermediate

Acyl-enzyme

Acyl-enzyme

Tetrahedralintermediate

OHNN

O

CO H

CN R1

OR2

–

HO

NN

O

CO H

CR1

O

H

–

– +

NR2

H ONN

O

CO H

CR1

O

–

R2

NH

H

ONN

O

CO H

CR1

O

–

R2 NHH

H O2

ONN

O

CO H

CO R1

O

H

H–

ONN

O

CO H

CO R1

O

H

H

–

– +O

HNN

OC

O H

CO R1

O

H

–

COR1

O

H

OHNN

OC

O H–

CNH

R1

OR2

7

3

4

5

6

1

8

Figure 7.25 Peptide hydrolysis by chymotrypsin. The mechanism of peptide hydrolysisillustrates the principles of covalent and acid–base catalysis. The reaction proceeds in eightsteps: (1) substrate binding, (2) serine’s nucleophilic attack on the peptide carbonyl group,(3) collapse of the tetrahedral intermediate, (4) release of the amine component, (5) waterbinding, (6) water’s nucleophilic attack on the acyl-enzyme intermediate, (7) collapse of thetetrahedral intermediate; and (8) release of the carboxylic acid component. The dashedgreen lines represent hydrogen bonds.

repeats steps 2 through 4. Again acting as a general base catalyst, histidine 57 drawsa proton away from the water molecule. The resulting OH� ion attacks the car-bonyl carbon atom of the acyl group, forming a tetrahedral intermediate (step 6).This structure breaks down to form the carboxylic acid product (step 7). Finally,the release of the carboxylic acid product (step 8) readies the enzyme for anotherround of catalysis.

This mechanism accounts for all characteristics of chymotrypsin action exceptthe observed preference for cleaving the peptide bonds just past residues with large,hydrophobic side chains. Examination of the three-dimensional structure of chy-motrypsin with substrate analogs and enzyme inhibitors revealed the presence of adeep, relatively hydrophobic pocket, called the S1 pocket, into which the long,uncharged side chains of residues such as phenylalanine and tryptophan can fit. Thebinding of an appropriate side chain into this pocket positions the adjacent peptide bondinto the active site for cleavage (Figure 7.27). The specificity of chymotrypsin dependsalmost entirely on which amino acid is directly on the amino-terminal side of thepeptide bond to be cleaved.

SUMMARY

7.1 A Few Basic Catalytic Strategies Are Used by Many EnzymesAlthough the detailed catalytic mechanisms of enzymes vary, manyenzymes use one or more common strategies. They include: (1) covalentcatalysis, in which the enzyme becomes temporarily covalently modified,(2) general acid–base catalysis, in which some molecule other than wateraccepts or donates a proton; (3) metal ion catalysis functions in a variety ofways; and (4) catalysis by approximation, in which substrates are broughtinto proximity and oriented to facilitate the reaction.

7.2 Enzyme Activity Can Be Modulated by Temperature, pH, andInhibitory MoleculesSpecific small molecules or ions can inhibit even nonallosteric enzymes. Inirreversible inhibition, the inhibitor is covalently linked to the enzyme orbound so tightly that its dissociation from the enzyme is very slow. Cova-lent inhibitors provide a means of mapping the enzyme’s active site. Incontrast, reversible inhibition is characterized by a more-rapid equilibriumbetween enzyme and inhibitor. A competitive inhibitor prevents the sub-strate from binding to the active site. It reduces the reaction velocity bydiminishing the proportion of enzyme molecules that are bound to sub-strate. Competitive inhibition can be overcome by raising the substrate con-centration. In uncompetitive inhibition, the inhibitor combines only withthe enzyme–substrate complex. In noncompetitive inhibition, the inhibitordecreases the turnover number. Uncompetitive and noncompetitive inhi-bition cannot be overcome by raising the substrate concentration.

7.3 Chymotrypsin Illustrates Basic Principles of Catalysis and InhibitionThe cleavage of peptide bonds by chymotrypsin is initiated by the attack bya serine residue on the peptide carbonyl group. The attacking hydroxylgroup is activated by interaction with the imidazole group of a histidine

104

Ser 195

Ser 189

Met 192

Ser 217

Gly 216 Gly 226

Trp 215

Ser 190

Figure 7.27 The specificity pocket of chymotrypsin. Notice that many hydrophobic groupsline the deep specificity pocket. The structure of the pocket favors the binding of residueswith long hydrophobic side chains such as phenylalanine (shown in green). Also notice thatthe active-site serine residue (serine 195) is positioned to cleave the peptide backbonebetween the residue bound in the pocket and the next residue in the sequence. The keyamino acids that constitute the binding site are identified.

QUICK QUIZ 2 Using the Cleland representation (Figure 6.6),

show the reaction progress of thehydrolysis of the tetrapeptide Ala-Phe-Gly-Ala by chymotrypsin.

Key Terms

covalent catalysis (p. 91)general acid–base catalysis (p. 92)metal ion catalysis (p. 92)catalysis by approximation (p. 92)binding energy (p. 92)competitive inhibition (p. 94)

uncompetitive inhibition (p. 95)noncompetitive inhibition (p. 95)group-specific reagent (p. 97)affinity label (substrate analog) (p. 97)mechanism-based (suicide)

inhibition (p. 98)

transition-state analog (p. 98)covalent modification (p. 100)catalytic triad (p. 102)oxyanion hole (p. 103)

Answers to QUICK QUIZZES

1. Curve 1, no inhibition; curve 2, competitive inhibi-tion; curve 3, noncompetitive inhibition; curve 4, uncom-petitive inhibition.

E Δ E-acyl intermediate Δ E-acyl intermediate Δ E

(Tetrapeptide) (H2O) (Ala-Phe)

Enzyme Enzyme

2. Tetrapeptidesubstrate Gly-Ala H2O Ala-Phe

1. Keeping busy. Many isolated enzymes, if incubated at37°C, will be denatured. However, if the enzymes are incu-bated at 37°C in the presence of substrate, the enzymes arecatalytically active. Explain this apparent paradox.

2. Controlled paralysis. Succinylcholine is a fast-acting,short-duration muscle relaxant that is used when a tube isinserted into a patient’s trachea or when a bronchoscope isused to examine the trachea and bronchi for signs of cancer.Within seconds of the administration of succinylcholine, thepatient experiences muscle paralysis and is placed on a respi-rator while the examination proceeds. Succinylcholine is acompetitive inhibitor of acetylcholinesterase, a nervous sys-tem enzyme, and this inhibition causes paralysis. However,succinylcholine is hydrolyzed by blood-serum cholinesterase,

which shows a broader substrate specificity than does the ner-vous system enzyme. Paralysis lasts until the succinylcholineis hydrolyzed by the serum cholinesterase, usually severalminutes later.(a) As a safety measure, serum cholinesterase is measuredbefore the examination takes place. Explain why this mea-surement is good idea.(b) What would happen to the patient if the serumcholinesterase activity were only 10 units of activity per literrather than the normal activity of about 80 units?(c) Some patients have a mutant form of the serumcholinesterase that displays a KM of 10 mM, rather than thenormal 1.4 mM. What will be the effect of this mutation onthe patient?

Problems

residue, which is, in turn, linked to an aspartate residue. This Ser-His-Aspcatalytic triad generates a powerful nucleophile. The product of this initialreaction is a covalent intermediate formed by the enzyme and an acyl groupderived from the bound substrate. The hydrolysis of this acyl-enzyme inter-mediate completes the cleavage process. The tetrahedral intermediates forthese reactions have a negative charge on the peptide carbonyl oxygen atom.This negative charge is stabilized by interactions with peptide NH groupsin a region on the enzyme termed the oxyanion hole.

105Problems

106 7 Mechanisms and Inhibitors

3. Mode of inhibition. The kinetics of an enzyme are mea-sured as a function of substrate concentration in the pres-ence and in the absence of 2 mM inhibitor (I).

Velocity (�mol minute�1)

[S] (�M) No inhibitor Inhibitor

3 10.4 4.15 14.5 6.4

10 22.5 11.330 33.8 22.690 40.5 33.8

(a) What are the values of Vmax and KM in the absence ofinhibitor? In its presence?(b) What type of inhibition is it?

4. A different mode. The kinetics of the enzyme consideredin problem 3 are measured in the presence of a differentinhibitor. The concentration of this inhibitor is 100 �M.

Velocity (�mol minute�1)

[S] (�M) No inhibitor Inhibitor

3 10.4 2.15 14.5 2.9

10 22.5 4.530 33.8 6.890 40.5 8.1

(a) What are the values of Vmax and KM in the presence ofthis inhibitor? Compare them with those obtained inproblem 3.(b) What type of inhibition is it?

5. Competing substrates. Suppose that two substrates, Aand B, compete for an enzyme. Derive an expression relat-ing the ratio of the rates of utilization of A and B, VA/VB, tothe concentrations of these substrates and their values ofkcat and KM. (Hint: Express VA as a function of kcat/KM forsubstrate A, and do the same for VB.) Is specificity deter-mined by KM alone?

6. Titration experiment. The effect of pH on the activity ofan enzyme was examined. At its active site, the enzyme hasan ionizable group that must be negatively charged inorder for substrate binding and catalysis to take place. Theionizable group has a pka of 6.0. The substrate is positivelycharged throughout the pH range of the experiment.

(a) Draw the V0-versus-pH curve when the substrate con-centration is much greater than the KM of the enzyme.(b) Draw the V0-versus-pH curve when the substrate con-centration is much less than the KM of the enzyme.(c) At which pH will the velocity equal one-half of the max-imal velocity attainable under these conditions?

7. No burst. Examination of the cleavage of the amide sub-strate, A, by chymotrypsin very early in the reaction revealsno burst. The reaction is monitored by noting the color pro-duced by the release of the amino part of the substrate(highlighted in orange). Why is no burst observed?

NO

O

HN

C

O

NH

C

O

H3C

A

CH2 H

C

8. Adding a charge. In chymotrypsin, a mutant was constructed with Ser 189, which is in the bottom of the sub-strate-specificity pocket, changed to Asp. What effect wouldyou predict for this Ser 189 S Asp 189 mutation?

9. Say no to cannibalism. If chymotrypsin is such an effec-tive protease, why doesn’t it digest itself?

10. Variations on a theme. Recall that TPCK, an affinity label,inactivates chymotrypsin by covalently binding to histidine57. Trypsin is a protease very similar to chymotrypsin, exceptthat it hydrolyzes peptide bonds on the carboxyl side of lysineor arginine.

(a) Name an affinity-labeling agent for trypsin.(b) How would you test the agent’s specificity?

E- + S+ Δ E-S+ ¡ E- + P+

+H+

EH

Δ

Selected readings for this chapter can be found onlineat www.whfreeman.com/Tymoczko