Voet D., Voet Ju.G. Biochemistry

Подождите немного. Документ загружается.

b. Certain Amino Acid Side Chains and Coenzymes

Can Serve as Covalent Catalysts

Enzymes commonly employ covalent catalytic mecha-

nisms as is indicated by the large variety of covalently

linked enzyme–substrate reaction intermediates that have

been isolated. For example, the enzymatic decarboxylation

of acetoacetate proceeds, much as described above,

through Schiff base formation with an enzyme Lys

residue’s ε-amino group. The covalent intermediate, in this

case, has been isolated through NaBH

reduction of its

imine bond to an amine, thereby irreversibly inhibiting the

enzyme. Other enzyme functional groups that participate

in covalent catalysis include the imidazole moiety of His,

the thiol group of Cys, the carboxyl function of Asp, and

the hydroxyl group of Ser. In addition, several coenzymes,

most notably thiamine pyrophosphate (Section 17-3Ba)

and pyridoxal phosphate (Section 26-1Aa), function in as-

sociation with their apoenzymes mainly as covalent cata-

lysts.

C. Metal Ion Catalysis

Nearly one-third of all known enzymes require the presence

of metal ions for catalytic activity. There are two classes of

metal ion–requiring enzymes that are distinguished by the

strengths of their ion–protein interactions:

1. Metalloenzymes contain tightly bound metal ions,

most commonly transition metal ions such as Fe

2

,Fe

3

2

,Zn

2

,Mn

2

, or Co

3

2. Metal-activated enzymes loosely bind metal ions

from solution, usually the alkali and alkaline earth metal

ions Na



,Mg

2

, or Ca

2

Metal ions participate in the catalytic process in three ma-

jor ways:

1. By binding to substrates so as to orient them prop-

erly for reaction.

2. By mediating oxidation–reduction reactions through

reversible changes in the metal ion’s oxidation state.

3. By electrostatically stabilizing or shielding negative

charges.

In this section we shall be mainly concerned with the third

aspect of metal ion catalysis. The other forms of enzyme-

mediated metal ion catalysis are considered in later chapters

in conjunction with discussions of specific enzyme mecha-

nisms.

a. Metal Ions Promote Catalysis through

Charge Stabilization

In many metal ion–catalyzed reactions, the metal ion

acts in much the same way as a proton to neutralize nega-

tive charge, that is, it acts as a Lewis acid.Yet metal ions are

often much more effective catalysts than protons because

metal ions can be present in high concentrations at neutral

pH’s and can have charges greater than 1. Metal ions have

therefore been dubbed “superacids.”

The decarboxylation of dimethyloxaloacetate, as cat-

alyzed by metal ions such as Cu

2

and Ni

2

, is a nonenzy-

matic example of catalysis by a metal ion:

Here the metal ion (M

n

), which is chelated by the di-

methyloxaloacetate, electrostatically stabilizes the devel-

oping enolate ion of the transition state. This mechanism is

supported by the observation that acetoacetate, which

cannot form such a chelate, is not subject to metal ion–

catalyzed decarboxylation. Most enzymes that decarboxy-

late oxaloacetate require a metal ion for activity.

b. Metal Ions Promote Nucleophilic Catalysis via

Water Ionization

A metal ion’s charge makes its bound water molecules

more acidic than free H

O and therefore a source of OH



ions even below neutral pH’s. For example, the water

molecule of (NH

)

3

O) ionizes according to the

reaction:

with a pK of 6.6, which is ⬃9 pH units below the pK of free

O. The resulting metal ion–bound hydroxyl group is a po-

tent nucleophile.

An instructive example of this phenomenon occurs in the

catalytic mechanism of carbonic anhydrase (Section 10-1C),

a widely occurring enzyme that catalyzes the reaction:

Carbonic anhydrase contains an essential Zn

2

ion that lies

at the bottom of an ⬃15-Å-deep active site cleft (Fig. 8-41),

where it is tetrahedrally coordinated by three evolutionar-

ily invariant His side chains and an O atom of either an

 H

O Δ HCO



 H



(NH

)

3

O) Δ (NH

)

3

(OH



)  H



–

CC CC

–

CC CH +

–

CC C

Dimethyloxaloacetate

Section 15-1. Catalytic Mechanisms 511

JWCL281_c15_506-556.qxd 2/19/10 9:27 PM Page 511

HCO

⫺

ion (Fig.15-5a) or a water molecule (Fig. 15-5b).The

enzyme has the following catalytic mechanism:

1. We begin with a water molecule bound to the protein

in the Zn

2⫹

ion’s fourth liganding position (Fig. 15-5b).This

2⫹

-polarized H

O ionizes in a process facilitated through

general base catalysis by His 64 in its “in” conformation.

Although His 64 is too far away from the Zn

2⫹

-bound wa-

ter to directly abstract its proton, these entities are linked

by two intervening water molecules to form a hydrogen

bonded network that is thought to act as a proton shuttle.

2. The resulting Zn

2⫹

-bound OH

⫺

ion nucleophilically

attacks the nearby enzymatically bound CO

, thereby con-

verting it to HCO

⫺

In doing so, the Zn

2⫹

-bound OH

⫺

group donates a hydro-

gen bond to Thr 199, which in turn donates a hydrogen

bond to Glu 106 (Fig. 15-5a). These interactions orient the

⫺

group with the optimal geometry (see below) for nu-

cleophilic attack on the substrate CO

3. The catalytic site is regenerated by the exchange of

the Zn

2⫹

-bound HCO

⫺

reaction product for H

O together

–

Im = imidazole

N HH

H H

His 64

–

Im = imidazole

His 64

512 Chapter 15. Enzymatic Catalysis

Figure 15-5 X-ray structures of human carbonic anhydrase.

(a) Its active site in complex with bicarbonate ion.The

polypeptide is shown in ribbon form (gold) with its side chains

shown in stick form colored according to atom type (C green,

N blue, and O red).The protein-bound Zn

2⫹

ion (cyan sphere) is

tetrahedrally liganded (gray bonds) by three invariant His side

chains and the HCO

⫺

ion, which is shown in ball-and-stick form.

The HCO

⫺

ion also interacts with the protein via van der Waals

contacts (dot surface colored according to atom type) and a

hydrogen bonded network (dashed gray lines) involving Thr 199

and Glu 106. [Based on an X-ray structure by K.K. Kannan,

Bhabha Atomic Research Center, Bombay, India. PDBid 1HCB.]

(b) The active site showing the proton shuttle through which His

64, acting as a general base, abstracts a proton from the

2⫹

-bound H

O to form an OH

⫺

ion.The polypeptide

backbone is shown in ribbon form (cyan), and its side chains and

several bound solvent molecules are shown in ball-and-stick

form with C black, N blue, and O red. The proton shuttle consists

of two water molecules that form a hydrogen bonded network

(dotted white lines) that bridges the Zn

2⫹

-bound OH

⫺

ion and

His 64 in its “in” conformation. On protonation, His 64 swings to

the “out” conformation. [Courtesy of David Christianson,

University of Pennsylvania.]

See Interactive Exercise 3

(a)

(b)

JWCL281_c15_506-556.qxd 10/19/10 7:19 AM Page 512

with the deprotonation of His 64. In the latter process, His

64 swings to its “out” conformation (Fig. 15-5b), which may

facilitate proton transfer to the bulk solvent.

c. Metal Ions Promote Reactions through

Charge Shielding

Another important enzymatic function of metal ions

is charge shielding. For example, the actual substrates of

kinases (phosphoryl-transfer enzymes utilizing ATP) are

2

–ATP complexes such as

rather than just ATP. Here, the Mg

2

ion’s role, in addition

to its orienting effect, is to shield electrostatically the nega-

tive charges of the phosphate groups. Otherwise, these

charges would tend to repel the electron pairs of attacking

nucleophiles, especially those with anionic character.

D. Electrostatic Catalysis

The binding of substrate generally excludes water from an

enzyme’s active site. The local dielectric constant of the ac-

tive site therefore resembles that in an organic solvent,

where electrostatic interactions are much stronger than

they are in aqueous solutions (Section 8-4A). The charge

distribution in a medium of low dielectric constant can

greatly influence chemical reactivity. Thus, as we have seen,

the pK’s of amino acid side chains in proteins may vary by

several units from their nominal values (Table 4-1) because

of the proximity of charged groups.

Although experimental evidence and theoretical analy-

ses on the subject are still sparse, there are mounting indica-

tions that the charge distributions about the active sites of

enzymes are arranged so as to stabilize the transition states

of the catalyzed reactions. Such a mode of rate enhance-

ment, which resembles the form of metal ion catalysis dis-

cussed above, is termed electrostatic catalysis. Moreover, in

several enzymes, these charge distributions apparently serve

to guide polar substrates toward their binding sites so that

the rates of these enzymatic reactions are greater than their

apparent diffusion-controlled limits (Section 14-2Bb).

E. Catalysis through Proximity and

Orientation Effects

Although enzymes employ catalytic mechanisms that re-

semble those of organic model reactions, they are far more

catalytically efficient than these models. Such efficiency

must arise from the specific physical conditions at enzyme

catalytic sites that promote the corresponding chemical re-

actions. The most obvious effects are proximity and orien-

tation: Reactants must come together with the proper spa-

tial relationship for a reaction to occur. For example, in the

Adenine Ribose O

OOO

PPPOOO

–

bimolecular reaction of imidazole with p-nitrophenyl-

acetate,

the progress of the reaction is conveniently monitored by

the appearance of the intensely yellow p-nitrophenolate ion:

[15.4]

where phenyl. Here k¿

, the pseudo-first-order rate con-

stant, is 0.0018 s

1

when [imidazole]  1M. However, for

the intramolecular reaction

the first-order rate constant k

 0.043 s

1

; that is, k

 24

k¿

. Thus, when the 1M imidazole catalyst is covalently at-

tached to the reactant, it is 24-fold more effective than

when it is free in solution; that is, the imidazole group in the

intramolecular reaction behaves as if its concentration is

24M. This rate enhancement has contributions from both

proximity and orientation.

a. Proximity Alone Contributes Relatively

Little to Catalysis

Let us make a rough calculation as to how the rate of a

reaction is affected purely by the proximity of its reacting

groups. Following Daniel Koshland’s treatment, we shall

make several reasonable assumptions:

1. Reactant species, that is, functional groups, are about

the size of water molecules.

2. Each reactant species in solution has 12 nearest-

neighbor molecules, as do packed spheres of identical size.

C O

NO

–

 k¿

[p-NO

Ac]

d[p-NO

O



]

 k

[imidazole] [p-NO

Ac]

p-Nitrophenylacetate

Imidazole

p-Nitrophenolate

N-Acetylimidazolium

(p-NO

␾Ac)

(p-NO

␾O

ⴚ

)

Section 15-1. Catalytic Mechanisms 513

JWCL281_c15_506-556.qxd 2/19/10 9:27 PM Page 513

3. Chemical reactions occur only between reactants

that are in contact.

4. The reactant concentration in solution is low enough

so that the probability of any reactant species being in si-

multaneous contact with more than one other reactant

molecule is negligible.

Then the reaction:

obeys the second-order rate equation

[15.5]

where [A, B]

pairs

is the concentration of contacting mole-

cules of A and B. The value of this quantity is

[15.6]

since there are 12 ways that A can be in contact with B, and

[A]/55.5M is the fraction of sites occupied by A in water so-

lution ([H

O]  55.5M in dilute aqueous solutions) and

hence the probability that a molecule of B will be next to

one of A. Combining Eqs. [15.5] and [15.6] yields

[15.7]

Thus, in the absence of other effects, this model predicts

that for the intramolecular reaction,

AB AB

v  k

55.5

b[A, B]

pairs

 4.6k

[A, B]

pairs

[A, B]

pairs



12[A] [B]

55.5M

v 

d[A¬B]

 k

[A][B]  k

[A, B]

pairs

A  B

A¬B

 4.6k

, which is a rather small rate enhancement. Fac-

tors that will increase this value other than proximity alone

clearly must be considered.

b. Properly Orienting Reactants and Arresting Their

Relative Motions Can Result in Large Catalytic

Rate Enhancements

The foregoing theory is, of course, quite simple. For ex-

ample, it does not take into account the relative orienta-

tions of the reacting molecules. Yet molecules are not

equally reactive in all directions as Koshland’s simple the-

ory assumes. Rather, they react most readily only if they

have the proper relative orientation. For example, in an S

(bimolecular nucleophilic substitution) reaction, the in-

coming nucleophile optimally attacks its target C atom

along the direction opposite to that of the bond to the leav-

ing group (Fig. 15-6). The approaches of reacting atoms

along a trajectory that deviates by as little as 10° from this

optimum direction can reduce the reaction rate by as much

as a factor of ⬃100. In a related phenomenon, a molecule

may be maximally reactive only when it assumes a confor-

mation that aligns its various orbitals in a way that minimizes

the electronic energy of its transition state, an effect termed

stereoelectronic assistance or stereoelectronic control.

Another effect that we have neglected in our treatment

of proximity is that of motions of the reacting groups with

respect to one another.Yet, in the transition state complex,

the reacting groups have little relative motion. In fact, as

Thomas Bruice demonstrated, the rates of intramolecular

reactions are greatly increased by arresting a molecule’s in-

ternal motions in a way that increases the mole fraction of

the reacting groups that are in a conformation which can

514 Chapter 15. Enzymatic Catalysis

R′

R′′

–

R′

–

R′′

–

R′

R′′

–p hybridization at carbon

Figure 15-6 The geometry of an S

2 reaction. The attacking

nucleophile, Y



, must approach the tetrahedrally coordinated

and hence sp

-hybridized C atom along the direction opposite

that of its bond to the leaving group, X, a process called backside

attack. In the transition state of the reaction, the C atom

becomes trigonal bipyramidally coordinated and hence sp

–p

hybridized, with the p orbital (blue) forming partial bonds to X

and Y. The three sp

orbitals form bonds to the C atom’s three

other substituents (R, R¿, and R–), which have shifted their

positions into the plane perpendicular to the axis

(curved arrows).Any deviation from this optimal geometry

would increase the free energy of the transition state, G

‡

, and

hence reduce the rate of the reaction (Eq. [14.15]). The transition

state then decomposes to products in which the R, R¿, and R–

have inverted their positions about the C atom, which has

rehybridized to sp

, and X



has been released.

X¬C¬Y

JWCL281_c15_506-556.qxd 6/5/10 9:10 AM Page 514

enter the transition state (Table 15-1). Similarly, when an

enzyme brings two molecules together in a bimolecular re-

action, as William Jencks pointed out, not only does it in-

crease their proximity, but it freezes out their relative

translational and rotational motions (decreases their en-

tropy), thereby enhancing their reactivity. Theoretical stud-

ies by Bruice indicate that much of this rate enhancement

can arise from the enzymatic binding of substrates in a con-

formation that readily enters the transition state.

Enzymes, as we shall see in Sections 16-2 and 16-3, bind

substrates in a manner that both aligns and immobilizes

them so as to optimize their reactivities. The free energy re-

quired to do so is derived from the specific binding free en-

ergy of substrate to enzyme.

F. Catalysis by Preferential Transition State Binding

The rate enhancements effected by enzymes are often

greater than can be reasonably accounted for by the cat-

alytic mechanisms so far discussed. However, we have not

yet considered one of the most important mechanisms of

enzymatic catalysis: the binding of the transition state to an

enzyme with greater affinity than the corresponding sub-

strates or products. When taken together with the previ-

ously described catalytic mechanisms, preferential transi-

tion state binding rationalizes the observed rates of

enzymatic reactions.

The original concept of transition state binding pro-

posed that enzymes mechanically strained their substrates

toward the transition state geometry through binding sites

into which undistorted substrates did not properly fit. This

so-called rack mechanism (in analogy with the medieval

torture device) was based on the extensive evidence for the

role of strain in promoting organic reactions. For example,

the rate of the reaction,

is 315 times faster when R is CH

rather than when it is H

because of the greater steric repulsions between the CH

groups and the reacting groups. Similarly, ring opening re-

actions are considerably more facile for strained rings such

as cyclopropane than for unstrained rings such as cyclo-

hexane. In either process, the strained reactant more closely

resembles the transition state of the reaction than does the

corresponding unstrained reactant. Thus, as was first sug-

gested by Linus Pauling and further amplified by Richard

Wolfenden and Gustav Lienhard, interactions that prefer-

entially bind the transition state increase its concentration

and therefore proportionally increase the reaction rate.

Let us quantitate this statement by considering the ki-

netic consequences of preferentially binding the transition

state of an enzymatically catalyzed reaction involving a sin-

gle substrate. The substrate S may react to form product P

COOH

Steric

strain

either spontaneously or through enzymatic catalysis:

The relationships between the various states of these two

reaction pathways are indicated in the following scheme:

where

are all association constants. Consequently,

[15.8]

According to transition state theory, Eqs. [14.7] and [14.14],

the rate of the uncatalyzed reaction can be expressed

[15.9]v

⫽ k

[S] ⫽ a

b[S

‡

] ⫽ a

‡

[S]

⫽

[S][ES

‡

]

‡

][ES]

⫽

‡

⫽

[E][S

‡

]

[E][S]

‡

⫽

[ES

‡

]

[ES]

⫽

[ES]

[E][S]

⫽

[ES

‡

]

[E][S

‡

]

ES Δ

‡

E ⫹ S Δ

‡

⫹ E

P ⫹ E

Section 15-1. Catalytic Mechanisms 515

Table 15-1 Relative Rates of Anhydride Formation for

Esters Possessing Different Degrees of

Motional Freedom in the Reaction:

Reactants

Relative Rate Constant

COO␾Br

COO

1.0

⬃1 ⫻ 10

⬃2.3 ⫻ 10

⬃8 ⫻ 10

Curved arrows indicate rotational degrees of freedom.

Source: Bruice, T.C. and Lightstone, F.C., Acc. Chem. Res. 32, 127 (1999).

JWCL281_c15_506-556.qxd 6/7/10 2:07 PM Page 515

Similarly, the rate of the enzymatically catalyzed reaction is

[15.10]

Therefore, combining Eqs. [15.8] to [15.10],

[15.11]

This equation indicates that the more tightly an enzyme

binds its reaction’s transition state (K

) relative to the sub-

strate (K

), the greater the rate of the catalyzed reaction (k

)

relative to that of the uncatalyzed reaction (k

); that is, catal-

ysis results from the preferential binding and therefore the

stabilization of the transition state (S

‡

) relative to that of the

substrate (S) (Fig. 15-7).

According to Eq. [14.15], the ratio of the rates of the cat-

alyzed versus the uncatalyzed reaction is expressed

[15.12]

A rate enhancement factor of 10

therefore requires that an

enzyme bind its transition state complex with 10

-fold

higher affinity than its substrate, which corresponds to a

34.2 kJ ⴢ mol

⫺1

stabilization at 25°C. This is roughly the free

energy of two hydrogen bonds. Consequently, the enzymatic

binding of a transition state (ES

‡

) by two hydrogen bonds

that cannot form in the Michaelis complex (ES) should result

in a rate enhancement of ⬃10

based on this effect alone.

It is commonly observed that the specificity of an en-

zyme is manifested by its turnover number (k

cat

) rather

than by its substrate-binding affinity. In other words, an en-

zyme binds poor substrates, which have a low reaction rate,

as well as or even better than good ones, which have a high

reaction rate. Such enzymes apparently use a good sub-

strate’s intrinsic binding energy to stabilize the correspon-

ding transition state; that is, a good substrate does not nec-

essarily bind to its enzyme with high affinity, but does so on

activation to the transition state.

⫽ exp[ (¢G

‡

⫺ ¢G

‡

)>RT]

⫽

‡

⫽

⫽ k

[ES] ⫽ a

b[ES

‡

] ⫽ a

‡

[ES]

a. Transition State Analogs Are Potent

Competitive Inhibitors

If an enzyme preferentially binds its transition state, then

it can be expected that transition state analogs, stable mol-

ecules that resemble S

‡

or one of its components, are potent

competitive inhibitors of the enzyme. For example, the reac-

tion catalyzed by proline racemase from Clostridium stick-

landii is thought to occur via a planar transition state:

Proline racemase is competitively inhibited by the planar

analogs of proline, pyrrole-2-carboxylate and ⌬-1-pyrroline-

2-carboxylate,

both of which bind to the enzyme with 160-fold greater

affinity than does proline. These compounds are there-

fore thought to be analogs of the transition state in the

proline racemase reaction. In contrast, tetrahydrofuran-

2-carboxylate,

which more closely resembles the tetrahedral structure of

proline, is not nearly as good an inhibitor as these com-

pounds. A 160-fold increase in binding affinity corre-

sponds, according to Eq. [15.12], to a 12.6 kJ ⴢ mol

⫺1

in-

crease in the free energy of binding. This quantity

presumably reflects the additional binding affinity that

proline racemase has for proline’s planar transition state

over that of the undistorted molecule.

Hundreds of transition state analogs for various enzy-

matic reactions have been reported. Some are naturally oc-

curring antibiotics. Others were designed to investigate the

mechanisms of particular enzymes and/or to act as specific

enzymatic inhibitors for therapeutic or agricultural use. In-

deed, as we discuss in Section 15-4C, the theory that en-

zymes bind transition states with higher affinity than sub-

strates has led to a rational basis for drug design based on

the understanding of specific enzyme reaction mechanisms.

COO

–

Tetrahydrofuran-2-carboxylate

Pyrrole-2-carboxylate ⌬-1-Pyrroline-2-carboxylate

COO

–

COO

–

COO

L-Proline

COO

D-Proline

COO

Planar transition

state

516 Chapter 15. Enzymatic Catalysis

ΔG

Reaction coordinate

E + S

E + P

Figure 15-7 Reaction coordinate diagrams for a hypothetical

enzymatically catalyzed reaction involving a single substrate

(blue) and the corresponding uncatalyzed reaction (red).

See the Animated Figures

JWCL281_c15_506-556.qxd 6/24/10 7:47 AM Page 516

2 LYSOZYME

In the following two sections, we shall investigate the cat-

alytic mechanisms of several well-characterized enzymes.

In doing so, we shall see how enzymes apply the catalytic

principles described in Section 15-1. You should note that

the great catalytic efficiency of enzymes arises from their si-

multaneous use of several of these catalytic mechanisms.

Lysozyme is an enzyme that destroys bacterial cell walls.

It does so, as we saw in Section 11-3Ba, by hydrolyzing the

(1 S 4) glycosidic linkages from N-acetylmuramic acid

(NAM) to N-acetylglucosamine (NAG) in the alternating

NAM–NAG polysaccharide component of cell wall pepti-

doglycans (Fig. 15-8). It likewise hydrolyzes (1 S 4)-linked

poly(NAG) (chitin), a cell wall component of most fungi.

Lysozyme occurs widely in the cells and secretions of ver-

tebrates, where it may function as a bactericidal agent.

However, the observation that few pathogenic bacteria are

susceptible to lysozyme alone has prompted the suggestion

that this enzyme mainly helps dispose of bacteria after they

have been killed by other means.

Hen egg white (HEW) lysozyme is the most widely

studied species of lysozyme and is one of the mechanisti-

cally best understood enzymes. It is a rather small (14.7 kD)

and readily available protein (an egg contains ⬃5 g of it),

whose single polypeptide chain consists of 129 amino acid

residues and is internally cross-linked by four disulfide

bonds (Fig. 15-9). HEW lysozyme catalyzes the hydrolysis

of its substrate at a rate that is ⬃10

-fold greater than that

of the uncatalyzed reaction.

A. Enzyme Structure

The elucidation of an enzyme’s mechanism of action re-

quires a knowledge of the structure of its enzyme–substrate

complex. This is because, even if the active site residues

have been identified through chemical, physical, and ge-

netic means, their three-dimensional arrangements relative

to the substrate as well as to each other must be known for

an understanding of how the enzyme works. However, an

enzyme binds its good substrates only transiently before it

catalyzes a reaction and releases the products. Conse-

quently, most of our knowledge of enzyme–substrate com-

plexes derives from X-ray studies of enzymes in complex

with inhibitors or poor substrates that remain stably bound

to the enzyme for the several hours that are usually re-

quired to measure a protein crystal’s X-ray diffraction in-

tensities (although techniques for measuring X-ray intensi-

ties in less than 1 s have been developed). The large

solvent-filled channels that occupy much of the volume of

most protein crystals (Section 8-3Aa) often permit the for-

mation of enzyme–inhibitor complexes by the diffusion of

inhibitor molecules into crystals of the native protein.

The X-ray structure of HEW lysozyme,which was eluci-

dated by David Phillips in 1965, was the second structure of

a protein and the first of an enzyme to be determined at

Section 15-2. Lysozyme 517

Figure 15-8 The alternating NAG–NAM polysaccharide component of bacterial cell walls.

The position of the lysozyme cleavage site is shown.

Figure 15-9 Primary structure of HEW lysozyme. The amino

acid residues that line the substrate-binding pocket are shown in

dark purple.

...

Lysozyme

cleavage

NAG NAM NAG NAM

H NH

C CH

CHCOO

–

H NH

C CH

H NH

C CH

H NH

C CH

CHCOO

–

COO

–

120

129

110

100

Lys

Val

Phe

Gly

Arg

Cys

Gly

Arg

Cys

Glu

Leu

Ala

Met

Lys

Arg

His

Gly

Leu

Asp

Gln

Ala

Trp

Ile

Asp

Val

Asn

Trp

Val

Cys

Leu

Ala

Lys

Phe

Asn

Thr

Gln

Ala

Arg

Asn

Thr

Asp

Gly

Ser

Thr

Tyr

Gly

Ile

Ser

Asp

Ile

Thr

Ala

Lys

Val

Ser

Val

Asn

Ala

Leu

Arg

Cys

Asp

Asn

Gly

Met

Gly

Arg

Ser

Arg

Trp

Asn

Thr

Pro

Ile

Thr

Phe

Glu

Ser

Asn

Asp

Leu

Gln

Asn

Trp

Leu

Asp

Gly

Asp

Val

Trp

Ala

Arg

Tyr

Thr

Ser

Leu

Arg

Gly

JWCL281_c15_506-556.qxd 2/19/10 9:27 PM Page 517

high resolution.The protein molecule is roughly ellipsoidal

in shape with dimensions 30 ⫻ 30 ⫻ 45 Å (Fig. 15-10). Its

most striking feature is a prominent cleft, the substrate-

binding site, that traverses one face of the molecule. The

polypeptide chain contains five helical segments as well as

a three-stranded antiparallel ␤ sheet that comprises much

of one wall of the binding cleft. As expected, most of the

nonpolar side chains are in the interior of the molecule, out

of contact with the aqueous solvent.

a. The Nature of the Binding Site

NAG oligosaccharides of less than five residues are but

very slowly hydrolyzed by HEW lysozyme (Table 15-2), al-

though these substrate analogs bind to the enzyme’s active

site and are thus its competitive inhibitors. The X-ray struc-

ture of the (NAG)

–lysozyme complex reveals that (NAG)

is bound at substrate subsites A, B, and C in Fig. 15-10.This

inhibitor associates with the enzyme through strong hydrogen

bonding interactions, some of which involve the acetamido

groups of residues A and C, as well as through close-fitting

hydrophobic contacts. In an example of induced-fit ligand

binding (Section 10-4C), there is a slight (⬃1 Å) closure of

lysozyme’s binding cleft on binding (NAG)

b. Lysozyme’s Catalytic Site Was Identified through

Model Building

(NAG)

takes several weeks to hydrolyze under the in-

fluence of lysozyme. It was therefore assumed that the

complex revealed by X-ray analysis is unproductive; that is,

the enzyme’s catalytic site occurs at neither the nor

the glycosidic bonds. [Presumably, the rare occasions

when (NAG)

is hydrolyzed occur when it binds produc-

tively at the catalytic site.]

In order to locate lysozyme’s catalytic site, Phillips used

model building to investigate how a larger substrate could bind

to the enzyme. Lysozyme’s active site cleft is long enough to

B¬C

A¬B

518 Chapter 15. Enzymatic Catalysis

Figure 15-10 X-ray structure of HEW lysozyme in complex

with (NAG)

. The protein is represented by its transparent

molecular surface with its polypeptide chain in ribbon form

colored in rainbow order from its N-terminus (blue) to its

C-terminus (red).The (NAG)

, which is drawn in stick form with

its sugar residues designated A, at its nonreducing end, through

F, at its reducing end, binds in a deep cleft in the enzyme surface.

Rings A, B, and C (colored according to atom type with C green,

N blue, and O red) are observed in the X-ray structure of the

complex of (NAG)

with lysozyme; the positions of rings D, E,

and F (C cyan, N blue, O red) were inferred by model building.

The side chains of lysozyme’s active site residues, Glu 35 and Asp

52, which are drawn in space-filling form (C yellow, O red),

catalyze the hydrolysis of the glycosidic bond between rings D

and E. [Based on an X-ray structure by David Phillips, Oxford

University. PDBid 1HEW.]

See Interactive Exercise 6 and

Kinemage Exercise 9.

JWCL281_c15_506-556.qxd 10/19/10 7:20 AM Page 518

accommodate (NAG)

, which the enzyme rapidly hydrolyzes

(Table 15-2). However, the fourth NAG residue (the D-ring in

Fig. 15-10) appeared unable to bind to the enzyme because its

C6 and O6 atoms too closely contact Glu 35, Trp 108, and the

acetamido group of the C-ring. This steric interference could

be relieved by distorting the glucose ring from its normal chair

conformation to that of a half-chair (Fig. 15-11). This distortion,

which renders atoms C1, C2, C5, and O5 of residue D coplanar,

moves the ¬C6H

OH group from its normal equatorial posi-

tion to an axial position where it makes no close contacts and can

hydrogen bond to the backbone carbonyl group of Gln 57 and

the amido group of Val 109 (Fig. 15-12). Continuing the model

building,Phillips found that the E- and F-rings apparently bind

to the enzyme without distortion and with a number of favor-

able hydrogen bonding and van der Waals contacts.

We are almost in a position to identify lysozyme’s cat-

alytic site. In the enzyme’s natural substrate, every second

Section 15-2. Lysozyme 519

Table 15-2 Rates of HEW Lysozyme-Catalyzed Hydrolysis

of Selected Oligosaccharide Substrate Analogs

Compound k

cat

⫺1

)

(NAG)

2.5 ⫻ 10

⫺8

(NAG)

8.3 ⫻ 10

⫺6

(NAG)

6.6 ⫻ 10

⫺5

(NAG)

0.033

(NAG)

0.25

(NAG–NAM)

0.5

Source: Imoto, T., Johnson, L.N., North,A.C.T., Phillips, D.C., and Rupley,

J.A., in Boyer, P.D. (Ed.), The Enzymes (3rd ed.),Vol. 7, p. 842, Academic

Press (1972).

Figure 15-11 Chair and half-chair conformations. Hexose

rings normally assume the chair conformation. It is postulated,

however, that binding by lysozyme distorts the D-ring into the

half-chair conformation such that atoms C1, C2, C5, and O5 are

coplanar.

See the Animated Figures

Figure 15-12 Interactions of lysozyme with its substrate. The

view is into the binding cleft with the heavier edges of the rings

facing the outside of the enzyme and the lighter ones against the

bottom of the cleft. [Illustration, Irving Geis. Image from the Irving

Geis Collection, Howard Hughes Medical Institute. Reprinted with

permission.] Based on an X-ray structure by David Phillips, Oxford

University, U.K. PDBid 4LYZ.]

See Kinemage Exercise 9

Chair conformation

Half-chair conformation

Ala

107

Asn

Trp

NAG

D ring in

half-chair

conformation

NAM

Lysozyme cuts

Asp 101

Gln

Phe

Glu

NAG

NAM

Asn

Arg

114

–

Val

109

Asp 52

–

Glu 35

JWCL281_c15_506-556.qxd 8/10/10 10:27 AM Page 519

residue is an NAM. Model building, however, indicated

that its lactyl side chain cannot be accommodated in the

binding subsites of either residues C or E. Hence,the NAM

residues must bind to the enzyme in subsites B, D, and F.

The observation that lysozyme hydrolyzes (1 S 4) linkages

from NAM to NAG indicates that bond cleavage occurs

either between rings B and C or rings D and E. Since (NAG)

is stably bound to but not cleaved by the enzyme while span-

ning subsites B and C, the probable cleavage site is between

rings D and E.This conclusion is supported by John Rupley’s

observation that lysozyme nearly quantitatively hydrolyzes

(NAG)

between the second and third residues from its re-

ducing terminus (the end with a free C1¬OH), just as is ex-

pected if the enzyme has six saccharide-binding subsites and

cleaves its bound substrate between rings D and E.

The bond that lysozyme cleaves was identified by carry-

ing out the lysozyme-catalyzed hydrolysis of (NAG)

O. The resulting product had

O bonded to the C1

atom of its newly liberated reducing terminus, thereby

demonstrating that bond cleavage occurs between C1 and

the bridge oxygen O1:

Thus, lysozyme catalyzes the hydrolysis of the C1¬O1

bond of a bound substrate’s D residue. Moreover,this reac-

tion occurs with retention of configuration, so that the D-

ring product remains the  anomer.

B. Catalytic Mechanism

It remains to identify lysozyme’s catalytic groups.The reac-

tion catalyzed by lysozyme, the hydrolysis of a glycoside, is

the conversion of an acetal to a hemiacetal. Nonenzymatic

acetal hydrolysis is an acid-catalyzed reaction that involves

the protonation of a reactant oxygen atom followed by

cleavage of its C¬O bond (Fig. 15-13). This results in the

formation of a resonance-stabilized carbocation that is

called an oxonium ion. To attain maximum orbital overlap,

and thus resonance stabilization, the oxonium ion’s R and

R¿ groups must be coplanar with its C, O, and H atoms

(stereoelectronic assistance). The oxonium ion then adds

water to yield the hemiacetal and regenerate the acid cata-

lyst. In searching for catalytic groups on an enzyme that

mediates acetal hydrolysis, we should therefore seek a

NAc

lysozyme

NAG NAM

NAG

NAM

reducing

end

()

potential acid catalyst and possibly a group that could fur-

ther stabilize an oxonium ion intermediate.

a. Glu 35 and Asp 52 Are Lysozyme’s

Catalytic Residues

The only functional groups in the immediate vicinity of

lysozyme’s reaction center that have the required catalytic

properties are the side chains of Glu 35 and Asp 52,

residues that are invariant in the family of lysozymes of

which HEW lysozyme is the prototype. These side chains,

which are disposed to either side of the (1 S 4) glycosidic

linkage to be cleaved (Fig. 15-10), have markedly different

environments. Asp 52 is surrounded by several conserved

polar residues with which it forms a complex hydrogen

bonded network. Asp 52 is therefore predicted to have a

normal pK; that is, it should be unprotonated and hence

negatively charged throughout the 3 to 8 pH range in which

lysozyme is catalytically active. In contrast, the carboxyl

group of Glu 35 is nestled in a predominantly nonpolar

pocket, where, as we discussed in Section 15-1D, it is likely to

remain protonated at unusually high pH’s for carboxyl

groups. Indeed, neutron diffraction studies, which provide

similar information to X-ray diffraction studies but also

reveal the positions of hydrogen atoms, indicate that Glu

35 is protonated at physiological pH. The closest ap-

proaches in the X-ray structures between the carboxyl O

atoms of both Asp 52 and Glu 35 and the C1¬O1 bond of

NAG D-ring are ⬃3 Å, which makes them the prime candi-

dates for electrostatic and acid catalysts, respectively.

520 Chapter 15. Enzymatic Catalysis

OR

H O R

R

+ H

OR

R

R

ROH

Acetal

Resonance-stabilized

carbocation (oxonium ion)

OR

H OH

Hemiacetal

Figure 15-13 Mechanism of the nonenzymatic acid-catalyzed

hydrolysis of an acetal to a hemiacetal. The reaction involves the

protonation of one of the acetal’s oxygen atoms followed by

cleavage of its bond to form an alcohol (R–OH) and a

resonance-stabilized carbocation (oxonium ion).The addition of

water to the oxonium ion forms the hemiacetal and regenerates

the H



catalyst. Note that the oxonium ion’s C, O, H, R, and R¿

atoms all lie in the same plane.

C¬O

JWCL281_c15_506-556.qxd 2/19/10 9:27 PM Page 520