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Chapter Enzymatic
15 Catalysis

1
Catalytic Mechanisms properties. We end with a discussion of how drugs are dis-
A. Acid–Base Catalysis covered and tested, a process that depends heavily on the
B. Covalent Catalysis principles of enzymology since many drug targets are
C. Metal Ion Catalysis enzymes. In doing so, we consider how therapeutically
D. Electrostatic Catalysis effective inhibitors of HIV-1 protease were discovered.
E. Catalysis through Proximity and Orientation Effects
F. Catalysis by Preferential Transition State Binding
2
Lysozyme 1
CATALYTIC MECHANISMS
A. Enzyme Structure
B. Catalytic Mechanism Catalysis is a process that increases the rate at which a
C. Testing the Phillips Mechanism reaction approaches equilibrium. Since, as we discussed in
3
Serine Proteases Section 14-1C, the rate of a reaction is a function of its free
A. Kinetics and Catalytic Groups energy of activation (
G ‡), a catalyst acts by lowering the
B. X-Ray Structures height of this kinetic barrier; that is, a catalyst stabilizes
C. Catalytic Mechanism the transition state with respect to the uncatalyzed reac-
D. Testing the Catalytic Mechanism tion. There is, in most cases, nothing unique about enzy-
E. Zymogens matic mechanisms of catalysis in comparison to nonenzy-
4
Drug Design matic mechanisms. What apparently make enzymes such
A. Techniques of Drug Discovery powerful catalysts are two related properties: their specificity
B. Introduction to Pharmacology of substrate binding combined with their optimal arrange-
C. HIV Protease and Its Inhibitors ment of catalytic groups. An enzyme’s arrangement of bind-
ing and catalytic groups is, of course, the product of eons
of evolution: Nature has had ample opportunity to fine-
tune the performances of most enzymes.
Enzymes, as we have seen, cause rate enhancements that
The types of catalytic mechanisms that enzymes employ
are orders of magnitude greater than those of the best
have been classified as:
chemical catalysts. Yet they operate under mild conditions
and are highly specific as to the identities of both their sub- 1. Acid–base catalysis.
strates and their products. These catalytic properties are 2. Covalent catalysis.
so remarkable that many nineteenth century scientists con-
cluded that enzymes have characteristics that are not 3. Metal ion catalysis.
shared by substances of nonliving origin. To this day, there 4. Electrostatic catalysis.
are few enzymes for which we understand in more than 5. Proximity and orientation effects.
cursory detail how they achieve their enormous rate ac-
6. Preferential binding of the transition state complex.
celerations. Nevertheless, it is now abundantly clear that
the catalytic mechanisms employed by enzymes are iden- In this section, we examine these various phenomena. In
tical to those used by chemical catalysts. Enzymes are doing so we shall frequently refer to the organic model
simply better designed. compounds that have been used to characterize these
In this chapter we consider the nature of enzymatic catalytic mechanisms.
catalysis. We begin by discussing the underlying principles
of chemical catalysis as elucidated through the study of or-
ganic reaction mechanisms. We then embark on a detailed
A. Acid–Base Catalysis
examination of the catalytic mechanisms of several of the General acid catalysis is a process in which partial proton
best characterized enzymes: lysozyme and the serine transfer from a Brønsted acid (a species that can donate
proteases. Their study should lead to an appreciation of protons; Section 2-2A) lowers the free energy of a reaction’s
the intracacies of these remarkably efficient catalysts as transition state. For example, an uncatalyzed keto–enol
well as of the experimental methods used to elucidate their tautomerization reaction occurs quite slowly as a result of

496
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Section 15–1. Catalytic Mechanisms 497

Keto Transition state Enol

R R R
δ–
(a) C O C O C O H
δ–
CH2 CH 2 CH2
δ+
H H

R R R
δ– δ+ δ– –
(b) C O + H A C O H A C O H + A
δ– H2O
CH2 CH 2 CH2
H+
δ+ –
H H H A + OH

R R R
δ–
(c) C O C O C O H
δ– +
CH2 CH 2 H CH2
δ+ +
H H +
+ H H
.. B
δ+ +..
B +
B B
FIGURE 15-1 Mechanisms of keto–enol tautomerization. (a) Uncatalyzed, (b) general acid
catalyzed, and (c) general base catalyzed.

the high energy of its carbanionlike transition state (Fig. In aqueous solvents, the initial rate of mutarotation of
15-1a). Proton donation to the oxygen atom (Fig. 15-1b),
-D-glucose, as monitored by polarimetry (Section 4-2A),
however, reduces the carbanion character of the transition is observed to follow the relationship:
state, thereby catalyzing the reaction. A reaction may also
be stimulated by general base catalysis if its rate is increased
by partial proton abstraction by a Bro/nsted base (a species d3
-D-glucose 4
that can combine with a proton; Fig. 15-1c). Some reactions v  kobs 3
-D-glucose4 [15.1]
dt
may be simultaneously subject to both processes: a con-
certed general acid–base catalyzed reaction.
where kobs is the reaction’s apparent first-order rate con-
a. Mutarotation Is Catalyzed by Acids and by Bases
stant. The mutarotation rate increases with the concentra-
The mutarotation of glucose provides an instructive
tions of general acids and general bases; they are thought
example of acid–base catalysis. Recall that a glucose
to catalyze mutarotation according to the mechanism:
molecule can assume either of two anomeric cyclic forms
through the intermediacy of its linear form (Section 11-1B):
CH2OH CH2OH H A
O O H A
H H H OH
H H O O O H B–
H
OH H OH H C C
HO OH HO H H
O H B–
H OH H OH

-D-Glucose -D-Glucose
-D-Glucose -D-Glucose
[
]20
D = 112.2 [
]20
D = 18.7

CH2OH
H OH H A–
H CH O O
OH H
HO CH O H B
H OH
Linear form Linear form
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498 Chapter 15. Enzymatic Catalysis

This model is consistent with the observation that in drolyze RNA to its component nucleotides. The isolation
aprotic solvents such as benzene, 2,3,4,6-O-tetramethyl- of 2,3-cyclic nucleotides from RNase A digests of RNA

-D-glucose (a less polar benzene-soluble analog) indicates that the enzyme mediates the following reaction
sequence:
CH2OCH3
H O H
H
OCH3 H O
H3CO OH –O 5
P O CH2 O
Base
H OCH3
O 4 1
2,3,4,6-O-Tetramethyl-
-D-glucose H H
H H
3 2
does not undergo mutarotation. Yet, the reaction is cat- O OH
alyzed by the addition of phenol, a weak benzene-soluble
–O P O CH2 O
acid, together with pyridine, a weak benzene-soluble base, RNA Base
according to the rate equation: O H H
v  k3 phenol 4 3pyridine 4 3tetramethyl-
-D-glucose4 [15.2] H H
O OH
Moreover, in the presence of
-pyridone, whose acid and
–O P O
base groups can rapidly interconvert between two tauto-
meric forms and are situated so that they can simultane- O
ously catalyze mutarotation,

-Pyridone
O
N N
–O P O CH2 O
O O Base
H
O H H
H H H
H H
O O O O
O O
C C HO CH2 O
P + Base
H H –O O H H
Glucose H H
2,3-Cyclic nucleotide
O OH
the reaction follows the rate law
–O P O
v  k¿ 3
-pyridone 4 3tetramethyl-
-D-glucose4 [15.3]
H2O O
where k  7000M  k. This increased rate constant indi- H+
cates that
-pyridone does, in fact, catalyze mutarotation
in a concerted fashion since 1M
-pyridone has the same
catalytic effect as impossibly high concentrations of phe- O
nol and pyridine (e.g., 70M phenol and 100M pyridine). –O P O CH2 O
Base
Many types of biochemically significant reactions are
O H H
susceptible to acid and/or base catalysis. These include the
H H
hydrolysis of peptides and esters, the reactions of phosphate
groups, tautomerizations, and additions to carbonyl groups. O OH
The side chains of the amino acid residues Asp, Glu, His, –O P O
Cys, Tyr, and Lys have pK’s in or near the physiological pH
range (Table 4-1) which, we shall see, permits them to act O–
in the enzymatic capacity of general acid and/or base cata-
The RNase A reaction exhibits a pH rate profile that peaks
lysts in analogy with known organic mechanisms. Indeed,
near pH 6 (Fig. 15-2). Analysis of this curve (Section 14-4),
the ability of enzymes to arrange several catalytic groups
together with chemical derivatization and X-ray studies,
about their substrates makes concerted acid–base catalysis
indicates that RNase A has two essential His residues, His
a common enzymatic mechanism.
12 and His 119, which act in a concerted manner as general
acid and base catalysts (the structure of RNase A is
b. The RNase A Reaction Incorporates General
sketched in Fig. 9-2). Evidently, the RNase A reaction is a
Acid–Base Catalysis
two-step process (Fig. 15-3):
Bovine pancreatic ribonuclease A (RNase A) provides
an illuminating example of enzymatically mediated general 1. His 12, acting as a general base, abstracts a proton
acid–base catalysis. This digestive enzyme functions to hy- from an RNA 2-OH group, thereby promoting its nucleo-
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Section 15–1. Catalytic Mechanisms 499

FIGURE 15-2 The pH dependence of V¿max  K¿M in the RNase

A–catalyzed hydrolysis of cytidine-2,3-cyclic phosphate.
V¿max K¿M is given in units of M1  s1. Analysis of this curve 5
(Section 14-4) suggests the catalytic participation of groups with
pK’s of 5.4 and 6.4. [After del Rosario, E.J. and Hammes, G.G.,
Biochemistry 8, 1887 (1969).]
V'max
log 3
K'M

pKE1 pKE2
1

4 5 6 7 8 9
pH

...
...

2′,3′-Cyclic nucleotide

O O
RNA
–O 5 –O P O CH2 O
P O CH2 O Base Base
4
H H
1 O H H
O
H H H H
N NH
3 2 H2O
O O H O O H N+ NH
His 12 1
P
–O P O CH2 O Base –O O
O H H O H
H H
HO CH2 O Base H
O OH

S
H H H N
–O P O H H
N
+ N
His 119 O O OH
H
N –O
...

H P O

O
...

2
...

O
–O P O CH2 O Base
O H H
H H

O O H SN NH
–O P O

O H

N+

N
FIGURE 15-3 The bovine pancreatic RNase A–catalyzed H
hydrolysis of RNA is a two-step process with the intermediate
formation of a 2,3-cyclic nucleotide.
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500 Chapter 15. Enzymatic Catalysis

philic attack on the adjacent phosphorus atom while His 1. The nucleophilic reaction between the catalyst and
119, acting as a general acid, promotes bond scission by the substrate to form a covalent bond.
protonating the leaving group. 2. The withdrawal of electrons from the reaction cen-
2. The 2,3-cyclic intermediate is hydrolyzed through ter by the now electrophilic catalyst.
what is essentially the reverse of the first step in which wa- 3. The elimination of the catalyst, a reaction that is es-
ter replaces the leaving group. Thus His 12 acts as a gen- sentially the reverse of stage 1.
eral acid and His 119 as a general base to yield the hy-
drolyzed RNA and the enzyme in its original state. Reaction mechanisms are somewhat arbitrarily classified
as occurring with either nucleophilic catalysis or electro-
philic catalysis depending on which of these effects pro-
B. Covalent Catalysis vides the greater driving force for the reaction, that is,
Covalent catalysis involves rate acceleration through the which catalyzes its rate-determining step. The primary
transient formation of a catalyst–substrate covalent bond. amine–catalyzed decarboxylation of acetoacetate is clearly
The decarboxylation of acetoacetate, as chemically cat- an electrophilically catalyzed reaction since its nucleophilic
alyzed by primary amines, is an example of such a process phase, Schiff base formation, is not its rate-determining
(Fig. 15-4). In the first stage of this reaction, the amine nu- step. In other covalently catalyzed reactions, however, the
cleophilically attacks the carbonyl group of acetoacetate to nucleophilic phase may be rate determining.
form a Schiff base (imine bond). The nucleophilicity of a substance is closely related to
its basicity. Indeed, the mechanism of nucleophilic catalysis
H H H resembles that of general base catalysis except that, instead
+ –
N + C O N C OH N C + OH of abstracting a proton from the substrate, the catalyst
nucleophilically attacks it so as to form a covalent bond.
H
Schiff Consequently, if covalent bond formation is the rate-
B H A base determining step of a covalently catalyzed reaction, the
reaction rate tends to increase with the covalent catalyst’s
The protonated nitrogen atom of the covalent intermediate basicity (pK).
then acts as an electron sink (Fig. 15-4, bottom) so as to re- An important aspect of covalent catalysis is that the
duce the otherwise high-energy enolate character of the tran- more stable the covalent bond formed, the less facilely it
sition state. The formation and decomposition of the Schiff will decompose in the final steps of a reaction. A good
base occur quite rapidly, so that these steps are not rate de- covalent catalyst must therefore combine the seemingly
termining in this reaction sequence. contradictory properties of high nucleophilicity and the
ability to form a good leaving group, that is, to easily
a. Covalent Catalysis Has Both Nucelophilic and reverse the bond formation step. Groups with high polar-
Electrophilic Stages izabilities (highly mobile electrons), such as imidazole and
As the preceding example indicates, covalent catalysis thiol functions, have these properties and hence make good
may be conceptually decomposed into three stages: covalent catalysts.

CO2 +
–O H
O O O

CH3 C CH2 C CH3 C CH2 CH3 C CH3

–
O

Acetoacetate Enolate Acetone

RNH2
 RNH2
OH 
OH

R H R H R H
 CO2 .. H+ 
N O N N

CH3 C CH2 C CH3 C CH2 CH3 C CH3 FIGURE 15-4 The decarboxylation of
– acetoacetate. The uncatalyzed reaction
O
mechanism is shown at the top and the
Schiff base reaction mechanism as catalyzed by
(imine) primary amines is shown at the bottom.
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Section 15–1. Catalytic Mechanisms 501

b. Certain Amino Acid Side Chains and Coenzymes The decarboxylation of dimethyloxaloacetate, as cat-
Can Serve as Covalent Catalysts alyzed by metal ions such as Cu2 and Ni2 , is a nonenzy-
Enzymes commonly employ covalent catalytic mecha- matic example of catalysis by a metal ion:
nisms as is indicated by the large variety of covalently
linked enzyme–substrate reaction intermediates that have M n+
been isolated. For example, the enzymatic decarboxylation –O O CH3 O
of acetoacetate proceeds, much as described above,
through Schiff base formation with an enzyme Lys residue’s C C C C
e-amino group. The covalent intermediate, in this case, has O CH3 O–
been isolated through NaBH4 reduction of its imine bond
Dimethyloxaloacetate
to an amine, thereby irreversibly inhibiting the enzyme.
Other enzyme functional groups that participate in cova-
lent catalysis include the imidazole moiety of His, the thiol CO2
group of Cys, the carboxyl function of Asp, and the hy-
droxyl group of Ser. In addition, several coenzymes, most
M n+
notably thiamine pyrophosphate (Section 17-3B) and
pyridoxal phosphate (Section 26-1A), function in associa- –O O– CH3
tion with their apoenzymes mainly as covalent catalysts. C C C
O CH3
C. Metal Ion Catalysis
Nearly one-third of all known enzymes require the presence H+
of metal ions for catalytic activity. There are two classes of
metal ion–requiring enzymes that are distinguished by the –O O CH3
strengths of their ion–protein interactions:
C C CH + Mn+
1. Metalloenzymes contain tightly bound metal ions, O CH3
most commonly transition metal ions such as Fe2 , Fe3 ,
Cu2 , Zn2 , Mn2 , or Co3 .
Here the metal ion (Mn ), which is chelated by the di-
2. Metal-activated enzymes loosely bind metal ions methyloxaloacetate, electrostatically stabilizes the devel-
from solution, usually the alkali and alkaline earth metal oping enolate ion of the transition state. This mechanism
ions Na , K , Mg2 , or Ca2 . is supported by the observation that acetoacetate, which
cannot form such a chelate, is not subject to metal ion–
Metal ions participate in the catalytic process in three
catalyzed decarboxylation. Most enzymes that decarboxy-
major ways:
late oxaloacetate require a metal ion for activity.
1. By binding to substrates so as to orient them prop-
b. Metal Ions Promote Nucleophilic Catalysis
erly for reaction.
via Water Ionization
2. By mediating oxidation–reduction reactions through A metal ion’s charge makes its bound water molecules
reversible changes in the metal ion’s oxidation state. more acidic than free H2O and therefore a source of OH
3. By electrostatically stabilizing or shielding negative ions even below neutral pH’s. For example, the water mol-
charges. ecule of (NH3)5Co3 (H2O) ionizes according to the reac-
tion:
1NH3 2 5Co3 1H2O2 ∆ 1NH3 2 5Co3 1OH 2 H
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 with a pK of 6.6, which is
9 pH units below the pK of
later chapters in conjunction with discussions of specific free H2O. The resulting metal ion–bound hydroxyl group
enzyme mechanisms. is a potent nucleophile.
An instructive example of this phenomenon occurs in
a. Metal Ions Promote Catalysis through the catalytic mechanism of carbonic anhydrase (Section
Charge Stabilization 10-1C), a widely occurring enzyme that catalyzes the
In many metal ion–catalyzed reactions, the metal ion reaction:
acts in much the same way as a proton to neutralize neg-
CO2 H 2O ∆ HCO3 H
ative charge, that is, it acts as a Lewis acid. Yet metal ions
are often much more effective catalysts than protons because Carbonic anhydrase contains an essential Zn2 ion that lies
metal ions can be present in high concentrations at neutral at the bottom of an
15-Å-deep active site cleft (Fig. 8-41),
pH’s and can have charges greater than 1. Metal ions have where it is tetrahedrally coordinated by three evolution-
therefore been dubbed “superacids.” arily invariant His side chains and an O atom of either an
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502 Chapter 15. Enzymatic Catalysis

HCO3 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 Zn2
ion’s fourth liganding position (Fig. 15-5b). This
Zn2
-polarized H2O 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 Zn2
-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.

H H H
H N N H O H O H O
Im Zn2+ Im
(a) Im

His 64

H H H
H N N H O H O H O–
+
Im Zn2+ Im

Im

His 64
Im = imidazole

2. The resulting Zn2
-bound OH ion nucleophilically

attacks the nearby enzymatically bound CO2, thereby con-
verting it to HCO3 .

Im O
2+
Im Zn O– + C
(b) Im H O
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
Im O
shown in stick form colored according to atom type (C green,
N blue, and O red). The protein-bound Zn2
ion (cyan sphere) Im Zn2+ O C
is tetrahedally liganded (gray bonds) by three invariant His
Im O–
side chains and the HCO3 ion, which is shown in ball-and-stick H
form. The HCO3 ion also interacts with the protein via van der H2O
Waals contacts (dot surface colored according to atom type) and
a hydrogen bonded network (dashed gray lines) involving Thr Im O
199 and Glu 106. [Based on an X-ray structure by K. K.
Kannan, Bhabha Atomic Research Center, Bombay, India. Im Zn
2+
O– + H+ + H O C
PDBid 1HCB.] (b) The active site showing the proton shuttle Im H O–
through which His 64, acting as a general base, abstracts a
proton from the Zn2
-bound H2O to form an OH ion. The Im = imidazole
polypeptide backbone is shown in ribbon form (cyan), and its
side chains and several bound solvent molecules are shown in In doing so, the Zn2
-bound OH group donates a hydro-
ball-and-stick form with C black, N blue, and O red. The gen bond to Thr 199, which in turn donates a hydrogen
proton shuttle consists of two water molecules that form a bond to Glu 106 (Fig. 15-5a). These interactions orient the
hydrogen bonded network (dotted white lines) that bridges the OH group with the optimal geometry (see below) for
Zn2
-bound OH ion and His 64 in its “in” conformation. On
nucleophilic attack on the substrate CO2.
protonation, His 64 swings to the “out” conformation.
[Courtesy of David Christianson, University of Pennsylvania.] 3. The catalytic site is regenerated by the exchange of
See the Interactive Exercises the Zn2
-bound HCO3 reaction product for H2O together
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Section 15–1. Catalytic Mechanisms 503

with the deprotonation of His 64. In the latter process, His the bimolecular reaction of imidazole with p-nitrophenyl-
64 swings to its “out” conformation (Fig. 15-5b), which may acetate,
facilitate proton transfer to the bulk solvent.
O
c. Metal Ions Promote Reactions through
CH3 C O NO2
Charge Shielding
Another important enzymatic function of metal ions is
charge shielding. For example, the actual substrates of p-Nitrophenylacetate
kinases (phosphoryl-transfer enzymes utilizing ATP) are O
N (p-NO2Ac)
Mg2 –ATP complexes such as
NH
Mg2+
O– O– O– Imidazole

Adenine Ribose O P O P O P O–
k1
O O O

rather than just ATP. Here, the Mg2 ion’s role, in addi- O
tion to its orienting effect, is to shield electrostatically the _
CH3 C + O NO2
negative charges of the phosphate groups. Otherwise, these
charges would tend to repel the electron pairs of attacking N
+
nucleophiles, especially those with anionic character. p-Nitrophenolate
NH (p-NO2O)

N-Acetylimidazolium
D. Electrostatic Catalysis
the progress of the reaction is conveniently monitored by
The binding of substrate generally excludes water from an
the appearance of the intensely yellow p-nitrophenolate
enzyme’s active site. The local dielectric constant of the ac-
ion:
tive site therefore resembles that in an organic solvent,
where electrostatic interactions are much stronger than d3p-NO2
O 4
they are in aqueous solutions (Section 8-4A). The charge  k1 3 imidazole4 3p-NO2
Ac4 [15.4]
 k¿1 3p-NO2
Ac4
dt
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 where
 phenyl. Here k¿1, the pseudo-first-order rate
vary by several units from their nominal values (Table 4-1) constant, is 0.0018 s1 when [imidazole]  1M. However,
because of the proximity of charged groups. for the intramolecular reaction
Although experimental evidence and theoretical analy-
O O
ses on the subject are still sparse, there are mounting indi-
cations that the charge distributions about the active sites of C O
NO2 C
k2
enzymes are arranged so as to stabilize the transition states + –O
NO2
N N+
of the catalyzed reactions. Such a mode of rate enhance-
ment, which resembles the form of metal ion catalysis dis- NH NH
cussed above, is termed electrostatic catalysis. Moreover,
in several enzymes, these charge distributions apparently
the first-order rate constant k2  0.043 s1; that is, k2 
serve to guide polar substrates toward their binding sites so
24k¿1. Thus, when the 1M imidazole catalyst is covalently
that the rates of these enzymatic reactions are greater than
attached to the reactant, it is 24-fold more effective than
their apparent diffusion-controlled limits (Section 14-2B).
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
E. Catalysis through Proximity and proximity and orientation.
Orientation Effects
a. Proximity Alone Contributes Relatively Little
Although enzymes employ catalytic mechanisms that re-
to Catalysis
semble those of organic model reactions, they are far more
Let us make a rough calculation as to how the rate of
catalytically efficient than these models. Such efficiency
a reaction is affected purely by the proximity of its react-
must arise from the specific physical conditions at enzyme
ing groups. Following Daniel Koshland’s treatment, we
catalytic sites that promote the corresponding chemical re-
shall make several reasonable assumptions:
actions. The most obvious effects are proximity and
orientation: Reactants must come together with the proper 1. Reactant species, that is, functional groups, are
spatial relationship for a reaction to occur. For example, in about the size of water molecules.
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504 Chapter 15. Enzymatic Catalysis

2. Each reactant species in solution has 12 nearest- Thus, in the absence of other effects, this model predicts
neighbor molecules, as do packed spheres of identical size. that for the intramolecular reaction,
3. Chemical reactions occur only between reactants k2
that are in contact. A B A B
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 k2
4.6k1, which is a rather small rate enhancement.
molecule is negligible. Factors that will increase this value other than proximity
Then the reaction: alone clearly must be considered.
k1
A  B ¡ A¬ B b. Properly Orienting Reactants and Arresting Their
Relative Motions Can Result in Large Catalytic Rate
obeys the second-order rate equation Enhancements
The foregoing theory is, of course, quite simple. For ex-

k1 3A4 3B 4
k2 3A, B4 pairs
d3A¬ B4
v
[15.5] ample, it does not take into account the relative orienta-
dt tions of the reacting molecules. Yet molecules are not
where [A,B]pairs is the concentration of contacting mole- equally reactive in all directions as Koshland’s simple the-
cules of A and B. The value of this quantity is ory assumes. Rather, they react most readily only if they
have the proper relative orientation. For example, in an SN2
12 3A4 3B 4 (bimolecular nucleophilic substitution) reaction, the in-
3A, B4 pairs
[15.6] coming nucleophile optimally attacks its target C atom
55.5M
along the direction opposite to that of the bond to the leav-
since there are 12 ways that A can be in contact with B, ing group (Fig. 15-6). The approaches of reacting atoms
and [A]
55.5M is the fraction of sites occupied by A in wa- along a trajectory that deviates by as little as 10 from this
ter solution ([H2O]
55.5M in dilute aqueous solutions) optimum direction can reduce the reaction rate by as much
and hence the probability that a molecule of B will be next as a factor of 100. In a related phenomenon, a molecule
to one of A. Combining Eqs. [15.5] and [15.6] yields may be maximally reactive only when it assumes a con-
formation that aligns its various orbitals in a way that min-
v
k1a b3 A, B4 pairs
4.6k1 3A, B4 pairs
55.5 imizes the electronic energy of its transition state, an effect
[15.7]
12 termed stereoelectronic assistance.

δ–
X X X–

R R′′
R′′ R′
R C C
C R′
R′′
R
R′

Yδ
–

Y– Y
sp2–p hybridization at carbon

FIGURE 15-6 The geometry of an SN2 reaction. The attacking three other substituents (R, R¿, and R– ), which have shifted
nucleophile, Y, must approach the tetrahedrally coordinated their positions into the plane perpendicular to the X¬ C¬ Y
and hence sp3-hybridized C atom along the direction opposite axis (curved arrows). Any deviation from this optimal geometry
that of its bond to the leaving group, X, a process called would increase the free energy of the transition state, G‡, and
backside attack. In the transition state of the reaction, the C hence reduce the rate of the reaction (Eq. [14.15]). The
atom becomes trigonal bipyramidally coordinated and hence transition state then decomposes to products in which the R,
sp2 –p hybridized, with the p orbital (blue) forming partial bonds R, and R have inverted their positions about the C atom,
to X and Y. The three sp2 orbitals form bonds to the C atom’s which has rehybridized to sp3, and X has been released.
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Section 15–1. Catalytic Mechanisms 505

Another effect that we have neglected in our treatment TABLE 15-1 Relative Rates of Anhydride Formation for
of proximity is that of motions of the reacting groups with Esters Possessing Different Degrees of Motional Freedom
respect to one another. Yet, in the transition state complex, in the Reaction:
the reacting groups have little relative motion. In fact, as O O
Thomas Bruice demonstrated, the rates of intramolecular
R1 C O Br R1 C
reactions are greatly increased by arresting a molecule’s in-
ternal motions in a way that increases the mole fraction of + O + _O Br
_
the reacting groups that are in a conformation which can R2 C O R2 C
enter the transition state (Table 15-1). Similarly, when an O
O
enzyme brings two molecules together in a bimolecular
reaction, as William Jencks pointed out, not only does it Reactantsa Relative Rate Constant
increase their proximity, but it freezes out their relative
CH3COO
Br
translational and rotational motions (decreases their en-
+ 1.0
tropy), thereby enhancing their reactivity. Theoretical _
studies by Bruice indicate that much of this rate enhance- CH3COO
ment can arise from the enzymatic binding of substrates in COO
Br
a conformation that readily enters the transition state.
1  103
Enzymes, as we shall see in Sections 16-2 and 16-3, bind _
COO
substrates in a manner that both aligns and immobilizes COO
Br
them so as to optimize their reactivities. The free energy
2.3  105
_
required to do so is derived from the specific binding free COO
energy of substrate to enzyme. O
COO
Br
_
8  107
F. Catalysis by Preferential Transition COO
State Binding a
Curved arrows indicate rotational degrees of freedom.
The rate enhancements effected by enzymes are often Source: Bruice, T.C. and Lightstone, F.C., Acc. Chem. Res. 32, 127
greater than can be reasonably accounted for by the cat- (1999).
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- as cyclopropane than for unstrained rings such as cyclo-
ously described catalytic mechanisms, preferential transi- hexane. In either process, the strained reactant more closely
tion state binding rationalizes the observed rates of resembles the transition state of the reaction than does the
enzymatic reactions. corresponding unstrained reactant. Thus, as was first sug-
The original concept of transition state binding pro- gested by Linus Pauling and further amplified by Richard
posed that enzymes mechanically strained their substrates Wolfenden and Gustav Lienhard, interactions that prefer-
toward the transition state geometry through binding sites entially bind the transition state increase its concentration
into which undistorted substrates did not properly fit. This and therefore proportionally increase the reaction rate.
so-called rack mechanism (in analogy with the medieval Let us quantitate this statement by considering the ki-
torture device) was based on the extensive evidence for netic consequences of preferentially binding the transition
the role of strain in promoting organic reactions. For state of an enzymatically catalyzed reaction involving a sin-
example, the rate of the reaction, gle substrate. The substrate S may react to form product
P either spontaneously or through enzymatic catalysis:
kN
R R H H S ¡ P
CH2OH kE
C ES ¡ EP
O + H2O
Here kE and kN are the first-order rate constants for the
COOH C
catalyzed and uncatalyzed reactions, respectively. The
R Steric R O relationships between the various states of these two re-
strain action pathways are indicated in the following scheme:
‡
KN
E S ∆ S E ¡ P E
‡

is 315 times faster when R is CH3 rather than when it is H

∆

because of the greater steric repulsions between the CH3 KR KT

groups and the reacting groups. Similarly, ring opening re- ‡
KE
actions are considerably more facile for strained rings such ES ∆ ES‡ ¡ EP
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506 Chapter 15. Enzymatic Catalysis

where E+S

3ES 4 3ES‡ 4
KR  KT 
3E4 3S4 3 E4 3S‡ 4 ∆GN

‡ 3E4 3S‡ 4 ‡ 3ES‡ 4

KN  KE 
3E4 3S 4 3 ES4
and ES

are all association constants. Consequently, G

∆GE
KT 3S4 3ES‡ 4 ‡
KE E+S
 ‡  ‡
3S 4 3ES4
[15.8]
KR KN ES
E+P
According to transition state theory, Eqs. [14.7] and EP
[14.14], the rate of the uncatalyzed reaction can be ex-
pressed
Reaction coordinate

vN  kN 3S4  a b 3S‡ 4  a b K N 3 S4 [15.9]

kkBT kkBT ‡
FIGURE 15-7 Reaction coordinate diagrams for a hypothetical
h h enzymatically catalyzed reaction involving a single substrate
(blue) and the corresponding uncatalyzed reaction (red).
Similarly, the rate of the enzymatically catalyzed reaction is
See the Animated Figures

vE  kE 3ES 4  a b 3ES‡ 4  a bKE 3 ES4 [15.10]

kkBT kkBT ‡
h h

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

kE KE
‡
KT a. Transition State Analogs Are Competitive Inhibitors
 ‡  [15.11] If an enzyme preferentially binds its transition state, then
kN KN KR
it can be expected that transition state analogs, stable
This equation indicates that the more tightly an enzyme molecules that resemble S ‡ or one of its components, are
binds its reaction’s transition state (KT) relative to the sub- potent competitive inhibitors of the enzyme. For example,
strate (KR), the greater the rate of the catalyzed reaction (kE) the reaction catalyzed by proline racemase from
relative to that of the uncatalyzed reaction (kN); that is, Clostridium sticklandii is thought to occur via a planar tran-
catalysis results from the preferential binding and therefore sition state:
the stabilization of the transition state (S ‡) relative to that _
COO H
of the substrate (S) (Fig. 15-7).
According to Eq. [14.15], the ratio of the rates of the C C
N H N _
catalyzed versus the uncatalyzed reaction is expressed COO
+ +
H H H H
 exp3 1 ¢GN  ¢GE 2 RT 4
kE ‡ ‡
[15.12] L-Proline D-Proline
kN
_ _
A rate enhancement factor of 106 therefore requires that C COO
an enzyme bind its transition state complex with 106-fold N
higher affinity than its substrate, which corresponds to a H
34.2 kJ  mol1 stabilization at 25C. This is roughly the
free energy of two hydrogen bonds. Consequently, the en- Planar transition
state
zymatic binding of a transition state (ES ‡) by two hydro-
gen bonds that cannot form in the Michaelis complex (ES) Proline racemase is competitively inhibited by the planar
should result in a rate enhancement of
106 based on this analogs of proline, pyrrole-2-carboxylate and -1-pyrro-
effect alone. line-2-carboxylate,
It is commonly observed that the specificity of an en-
zyme is manifested by its turnover number (kcat) rather
than by its substrate-binding affinity. In other words, an COO– + COO–
enzyme binds poor substrates, which have a low reaction N N
rate, as well as or even better than good ones, which have H H
a high reaction rate. Such enzymes apparently use a good Pyrrole-2-carboxylate
-1-Pyrroline-2-carboxylate
substrate’s intrinsic binding energy to stabilize the corre-
sponding transition state; that is, a good substrate does not both of which bind to the enzyme with 160-fold greater
necessarily bind to its enzyme with high affinity, but does affinity than does proline. These compounds are therefore
so on activation to the transition state. thought to be analogs of the transition state in the proline
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Section 15–2. Lysozyme 507

Lysozyme
6CH2OH CH2OH cleavage CH2OH CH2OH
H 5 O H O H O H O
... H H H
O
H O...
4 1 O O
O OH H 1 H OH H H
H H H H
3 2
H NH C CH3 H NH C CH3 H NH C CH3 H NH C CH3
O O
O O O O
CH3CHCOO
– CH3CHCOO–

NAG NAM NAG NAM

FIGURE 15-8 The alternating NAG–NAM polysaccharide component of bacterial cell walls.
The position of the lysozyme cleavage site is shown.

racemase reaction. In contrast, tetrahydrofuran-2-car- susceptible to lysozyme alone has prompted the suggestion
boxylate, that this enzyme mainly helps dispose of bacteria after they
have been killed by other means.
COO– Hen egg white (HEW) lysozyme is the most widely
studied species of lysozyme and is one of the mechanisti-
O H
cally best understood enzymes. It is a rather small protein
Tetrahydrofuran-2-carboxylate (14.7 kD) whose single polypeptide chain consists of 129
amino acid residues and is internally cross-linked by four
which more closely resembles the tetrahedral structure of disulfide bonds (Fig. 15-9). HEW lysozyme catalyzes the
proline, is not nearly as good an inhibitor as these
compounds. A 160-fold increase in binding affinity corres-
ponds, according to Eq. [15.12], to a 12.6 kJ  mol1
increase in the free energy of binding. This quantity
presumably reflects the additional binding affinity that Leu
Asp Asn Tyr
Arg Gly Tyr Ser
Leu
Gly Gly
proline racemase has for proline’s planar transition state His
20 Asn
Trp
over that of the undistorted molecule. Arg
Ala Gln Val Asp Val
Trp Thr
Hundreds of transition state analogs for various enzy- Lys
120 Gly Cys 30
Ile S
matic reactions have been reported. Some are naturally Met Lys Ala
Ala
Arg Cys S
occurring antibiotics. Others were designed to investigate Gly
Cys 129 Ala
Ala Arg Arg
the mechanisms of particular enzymes and/or to act as spe- Ala S Leu Lys
10
Leu COO– Asn
cific enzymatic inhibitors for therapeutic or agricultural Glu S Phe
Arg
use. Indeed, as we discuss in Section 15-4C, the theory that Cys Glu
Trp
enzymes bind transition states with higher affinity than sub- Arg Val Ala Ser
Trp
strates has led to a rational basis for drug design based on 1
Gly Ala 110
Asn
Phe Asn
the understanding of specific enzyme reaction mechanisms. H3N+ Lys Val Phe
Met
Gly Arg Ser Asn
Asn Gly
Leu
Asp Pro Thr 40
2
LYSOZYME Gly
Cys 70 Thr
Gln
Asp Asn
In the following two sections, we shall investigate the cat- S Arg Ala
Ser 100 Ile
Thr
alytic mechanisms of several well-characterized enzymes. Val Pro
Gly
Asn Asn
In doing so, we shall see how enzymes apply the catalytic Ile Cys 80
principles described in Section 15-1. You should note that S Asp Arg
Lys Ser S Cys
the great catalytic efficiency of enzymes arises from their si- Lys Ala
Asn
Trp
multaneous use of several of these catalytic mechanisms. S Leu
Thr
Ala Trp
Lysozyme is an enzyme that destroys bacterial cell walls. Asp
Cys Leu
Arg
It does so, as we saw in Section 11-3B, by hydrolyzing the Gly
Asn Ser
(1S 4) glycosidic linkages from N-acetylmuramic acid Ser
Ser 60 50 Ser
Val 90 Asn
(NAM) to N-acetylglucosamine (NAG) in the alternating Ser Asp Thr
Ala Thr Ile Ile
NAM–NAG polysaccharide component of cell wall pepti- Asp
doglycans (Fig. 15-8). It likewise hydrolyzes (1S4)-linked
Gln Tyr
Leu Ile Gly
poly(NAG) (chitin), a cell wall component of most fungi.
Lysozyme occurs widely in the cells and secretions of ver- FIGURE 15-9 Primary structure of HEW lysozyme. The amino
tebrates, where it may function as a bactericidal agent. acid residues that line the substrate-binding pocket are shown
However, the observation that few pathogenic bacteria are in dark purple.
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508 Chapter 15. Enzymatic Catalysis

hydrolysis of its substrate at a rate that is
108-fold greater ture of the (NAG)3 –lysozyme complex reveals that
than that of the uncatalyzed reaction. (NAG)3 is bound on the right side of the enzymatic bind-
ing cleft as drawn in Fig. 15-10a for substrate residues A,
B, and C. This inhibitor associates with the enzyme through
A. Enzyme Structure strong hydrogen bonding interactions, some of which in-
The elucidation of an enzyme’s mechanism of action re- volve the acetamido groups of residues A and C, as well
quires a knowledge of the structure of its enzyme–substrate as through close-fitting hydrophobic contacts. In an ex-
complex. This is because, even if the active site residues ample of induced-fit ligand binding (Section 10-4C), there
have been identified through chemical and physical means, is a slight (
1 Å) closure of lysozyme’s binding cleft on
their three-dimensional arrangements relative to the sub- binding (NAG)3.
strate as well as to each other must be known for an
understanding of how the enzyme works. However, an en- b. Lysozyme’s Catalytic Site Was Identified through
zyme binds its good substrates only transiently before it Model Building
catalyzes a reaction and releases the products. Conse- (NAG)3 takes several weeks to hydrolyze under the in-
quently, most of our knowledge of enzyme–substrate fluence of lysozyme. It is therefore presumed that the com-
complexes derives from X-ray studies of enzymes in com- plex revealed by X-ray analysis is unproductive; that is, the
plex with inhibitors or poor substrates that remain stably enzyme’s catalytic site occurs at neither the A¬ B nor the
bound to the enzyme for the several hours that are usually B¬ C bonds. [Presumably, the rare occasions when
required to measure a protein crystal’s X-ray diffraction (NAG)3 hydrolyzes occur when it binds productively at the
intensities (although techniques for measuring X-ray in- catalytic site.]
tensities in less than 1 s have been developed). The large In order to locate lysozyme’s catalytic site, Phillips used
solvent-filled channels that occupy much of the volume of model building to investigate how a larger substrate could
most protein crystals (Section 8-3A) often permit the for- bind to the enzyme. Lysozyme’s active site cleft is long
mation of enzyme–inhibitor complexes by the diffusion of enough to accommodate (NAG)6, which the enzyme
inhibitor molecules into crystals of the native protein. rapidly hydrolyzes (Table 15-2). However, the fourth NAG
The X-ray structure of HEW lysozyme, which was elu- residue (residue D in Fig. 15-10a) appeared unable to bind
cidated by David Phillips in 1965, was the second structure to the enzyme because its C6 and O6 atoms too closely
of a protein and the first of an enzyme to be determined contact Glu 35, Trp 108, and the acetamido group of NAG
at high resolution. The protein molecule is roughly ellip- residue C. This steric interference could be relieved by dis-
soidal in shape with dimensions 30  30  45 Å (Fig. torting the glucose ring from its normal chair conforma-
15-10). Its most striking feature is a prominent cleft, the
substrate-binding site, that traverses one face of the mole-
cule. The polypeptide chain forms five helical segments as
well as a three-stranded antiparallel  sheet that comprises
much of one wall of the binding cleft (Fig. 15-10b). As ex-
pected, most of the nonpolar side chains are in the interior
of the molecule, out of contact with the aqueous solvent.
FIGURE 15-10 (Opposite) X-Ray structure of HEW lysozyme.
a. The Nature of the Binding Site (a) The polypeptide chain is shown with a bound (NAG)6
NAG oligosaccharides of less than five residues are but substrate (green). The positions of the backbone C
atoms are
very slowly hydrolyzed by HEW lysozyme (Table 15-2) al- indicated together with those of the side chains that line the
substrate-binding site and form disulfide bonds. The substrate’s
though these substrate analogs bind to the enzyme’s active
sugar rings are designated A, at its nonreducing end (right),
site and are thus its competitive inhibitors. The X-ray struc- through F, at its reducing end (left). Lysozyme catalyzes the
hydrolysis of the glycosidic bond between residues D and E.
Rings A, B, and C are observed in the X-ray structure of the
complex of (NAG)3 with lysozyme; the positions of rings D, E,
TABLE 15-2 Rates of HEW Lysozyme-Catalyzed and F were inferred from model building studies. [Illustration,
Hydrolysis of Selected Oligosaccharide Substrate Analogs Irving Geis/Geis Archives Trust. Copyright Howard Hughes
Compound kcat (s
1) Medical Institute. Reproduced with permission.] (b) A ribbon
diagram of lysozyme highlighting the protein’s secondary
(NAG)2 2.5  108 structure and indicating the positions of its catalytically
(NAG)3 8.3  106 important side chains, Glu 35 and Asp 52 (red). (c) A
(NAG)4 6.6  105 computer-generated model showing the protein’s molecular
(NAG)5 0.033 envelope ( purple) and C
backbone (blue). The side chains of
the catalytic residues, Asp 52 (above) and Glu 35 (below), are
(NAG)6 0.25
colored yellow. Note the enzyme’s prominent substrate-binding
(NAG–NAM)3 0.5 cleft. [Courtesy of Arthur Olson, The Scripps Research
Source: Imoto, T., Johnson, L.N., North, A.C.T., Phillips, D.C., and Institute, La Jolla, California.] Parts a, b, and c have
Rupley, J.A., in Boyer, P.D. (Ed.), The Enzymes (3rd ed.), Vol. 7, p. 842, approximately the same orientation. See the Interactive
Academic Press (1972). Exercises and Kinemage Exercise 9
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Section 15–2. Lysozyme 509

(a)

(b) (c)

Asp 52

Glu 35

C
7884d_c15.qxd 3/24/03 1:01 PM Page 510 mac85 Mac 85:1st shift: 1268_tm:7884d:

510 Chapter 15. Enzymatic Catalysis

OH

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

which renders atoms C1, C2, C5, and O5 of residue D copla- HO CH2OH
nar, moves the ¬ C6H2OH group from its normal equa- O A
torial position to an axial position where it makes no close C NAG
O –O
contacts and can hydrogen bond to the backbone carbonyl H3C N
group of Gln 57 and the amido group of Val 109 (Fig. 15- C Asp 101
12). Continuing the model building, Phillips found that H O
residues E and F apparently bind to the enzyme without O
distortion and with a number of favorable hydrogen bond- O H
ing and van der Waals contacts. R O
We are almost in a position to identify lysozyme’s cat- CH2
alytic site. In the enzyme’s natural substrate, every second B NAM
H
residue is an NAM. Model building, however, indicated
that its lactyl side chain cannot be accommodated in the N O
C
binding subsites of either residues C or E. Hence, the NAM
H3C O
O
H Trp
NAG NAM
A B
NAG NAM
C D
NAG NAM
E F
( reducing
end ) Trp
62
CH2 O H N 63

N H O
C NAG
H Ala
residues must bind to the enzyme in subsites B, D, and F. O N H O C
107
C
The observation that lysozyme hydrolyzes (1S4) link- Asn CH3
O
ages from NAM to NAG implies that bond cleavage occurs 59 N H
O
either between residues B and C or between residues D Val
H N
and E. Since (NAG)3 is stably bound to but not cleaved R O 109
by the enzyme while spanning subsites B and C, the prob- D ring in CH2O
able cleavage site is between residues D and E. This con- half-chair O D Gln
NAM H O C
clusion is supported by John Rupley’s observation that conformation 57
C O O
lysozyme nearly quantitatively hydrolyzes (NAG)6 H3C N – C Asp 52
O
H Lysozyme cuts
NH2
O H O
Gln C
C
C4 57 O H Glu 35
O5 O CH2OH O
C5
Asn NH2 O E
44 C NAG
C
O
O H3C N

H
C2 Glu Phe
C3 C O O H O C
C1 35 34
Chair conformation R O O
CH2 H2N
C5 O Asn
F C
C4 C NAM 37
O
H3C N O
H2N
O5 H
+ NH
O H2N Arg
H 114
C2
C3 FIGURE 15-12 Interactions of lysozyme with its substrate. The
C1
Half-chair conformation view is into the binding cleft with the heavier edges of the rings
facing the outside of the enzyme and the lighter ones against
FIGURE 15-11 Chair and half-chair conformations. Hexose the bottom of the cleft. [Illustration, Irving Geis/Geis Archives
rings normally assume the chair conformation. It is postulated, Trust. Copyright Howard Hughes Medical Institute. Reproduced
however, that binding by lysozyme distorts the D-ring into the with permission. Based on an X-ray structure by David Phillips,
half-chair conformation such that atoms C1, C2, C5, and O5 are Oxford University, U.K. PDBid 4LYZ.] See Kinemage
coplanar. See the Animated Figures Exercise 9
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Section 15–2. Lysozyme 511

between the second and third residues from its reducing OR OR
terminus (the end with a free C1¬OH), just as is expected + +
H C O R + H H C O R
if the enzyme has six saccharide-binding subsites and
cleaves its bound substrate between residues D and E. R R H
The bond that lysozyme cleaves was identified by car- Acetal ROH
rying out the lysozyme-catalyzed hydrolysis of (NAG)3 in
H218O. The resulting product had 18O bonded to the C1 R R
+
atom of its newly liberated reducing terminus, thereby O O
demonstrating that bond cleavage occurs between C1 and +
C C
the bridge oxygen O1: H R H R
Resonance-stabilized
carbocation (oxonium ion)
H2O
CH2OH +
H H
O
H
C1 O1 C
H 4 OH OR

H C OH
NAc H
18 R
H2 O lysozyme
Hemiacetal
CH2OH FIGURE 15-13 Mechanism of the nonenzymatic acid-catalyzed
18
O OH H
hydrolysis of an acetal to a hemiacetal. The reaction involves
H the protonation of one of the acetal’s oxygen atoms followed by
C + C
H OH cleavage of its C¬ O bond to form an alcohol (ROH) and a
H HO resonance-stabilized carbocation (oxonium ion). The addition
NAc H 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.

Thus, lysozyme catalyzes the hydrolysis of the C1 ¬ O1

bond of a bound substrate’s D residue. Moreover, this
reaction 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 properties are the side chains of Glu 35 and Asp 52,
reaction catalyzed by lysozyme, the hydrolysis of a glyco- residues that are invariant in the family of lysozymes of
side, is the conversion of an acetal to a hemiacetal. which HEW lysozyme is the prototype. These side chains,
Nonenzymatic acetal hydrolysis is an acid-catalyzed reac- which are disposed to either side of the (1S 4) glycosidic
tion that involves the protonation of a reactant oxygen linkage to be cleaved (Fig. 15-10), have markedly different
atom followed by cleavage of its C ¬ O bond (Fig. 15-13). environments. Asp 52 is surrounded by several conserved
This results in the formation of a resonance-stabilized car- polar residues with which it forms a complex hydrogen
bocation that is called an oxonium ion. To attain maximum bonded network. Asp 52 is therefore predicted to have a
orbital overlap, and thus resonance stabilization, the normal pK; that is, it should be unprotonated and hence
oxonium ion’s R and R groups must be coplanar with its negatively charged throughout the 3 to 8 pH range in which
C, O, and H atoms (stereoelectronic assistance). The oxo- lysozyme is catalytically active. In contrast, the carboxyl
nium ion then adds water to yield the hemiacetal and re- group of Glu 35 is nestled in a predominantly nonpolar
generate the acid catalyst. In searching for catalytic groups pocket, where, as we discussed in Section 15-1D, it is likely
on an enzyme that mediates acetal hydrolysis, we should to remain protonated at unusually high pH’s for carboxyl
therefore seek a potential acid catalyst and possibly a groups. Indeed, neutron diffraction studies, which provide
group that could further stabilize an oxonium ion similar information to X-ray diffraction studies but also re-
intermediate. veal the positions of hydrogen atoms, indicate that Glu 35
is protonated at physiological pH’s. The closest approaches
a. Glu 35 and Asp 52 Are Lysozyme’s in the X-ray structures between the carboxyl O atoms of
Catalytic Residues both Asp 52 and Glu 35 and the C1 ¬ O1 bond of NAG
The only functional groups in the immediate vicinity of residue D are
3 Å, which makes them the prime candi-
lysozyme’s reaction center that have the required catalytic dates for electrostatic and acid catalysts, respectively.
7884d_c15.qxd 1/23/03 12:28 PM Page 512 mac18 mac18:df_169:7884D:

512 Chapter 15. Enzymatic Catalysis

b. The Phillips Mechanism

With much of the foregoing information, Phillips pos-
tulated the following enzymatic mechanism for lysozyme
(Fig. 15-14):
1. Lysozyme attaches to a bacterial cell wall by binding
Lysozyme,
to a hexasaccharide unit. In the process, residue D is dis- main chain Asp 52
torted toward the half-chair conformation in response to
the unfavorable contacts that its ¬ C6H2OH group would
otherwise make with the protein.
2. Glu 35 transfers its proton to the O1 of the D-ring, O– NAc
the only polar group in its vicinity (general acid catalysis). D
The C1 ¬ O1 bond is thereby cleaved, generating a reso-
nance-stabilized oxonium ion at C1.
O+
3. The ionized carboxyl group of Asp 52 acts to stabilize CH2OH
the developing oxonium ion through charge–charge inter-
actions (electrostatic catalysis). This carboxylate group O–
apparently cannot form a covalent bond with the substrate
because the observed
3 Å distance between C1 and a car- E H+
boxyl O atom of Asp 52 is much greater than the
1.4 Å
length of a C¬ O covalent bond [i.e., the reaction appears NAc OH–
CH2OH
to occur via an SN1 (unimolecular nucleophilic substitu-
tion) mechanism to yield an oxonium ion, not via a Glu 35
mechanism involving the transient formation of a C ¬ O
covalent bond to the enzyme; but see Section 15-2C]. The H+
bond cleavage reaction is facilitated by the strain in the
D-ring that distorts it to the planar half-chair conforma- Lysozyme,
tion. This is a result of the oxonium ion’s required pla- main chain
narity; that is, the initial binding conformation of the D-ring
H2O
resembles that of the reaction’s transition state (transition
state binding catalysis; Fig. 15-15).
FIGURE 15-14 The Phillips mechanism for the lysozyme
4. At this point, the enzyme releases the hydrolyzed
reaction. The cleavage of the glycosidic bond between the
E-ring with its attached polysaccharide (the leaving group), substrate D- and E-rings occurs through protonation of the
yielding a cationic, noncovalent, glycosyl–enzyme inter- bridge oxygen atom by Glu 35. The resulting D-ring oxonium
mediate. This oxonium ion subsequently adds H2O from ion is stabilized by the proximity of the Asp 52 carboxylate
solution in a reversal of the preceding steps to form prod- group and the enzyme-induced distortion of the D-ring. Once
uct and to reprotonate Glu 35. The reaction’s retention of the E-ring is released, H2O from solution provides both an
configuration is dictated by the shielding of one of the ox- OH that combines with the oxonium ion and an H that
onium ion’s faces by the enzymatic cleft. The enzyme then reprotonates Glu 35. NAc represents the N-acetylamino
releases the D-ring product with its attached saccharide, substituent at C2 of each glucose ring. See Kinemage
thereby completing the catalytic cycle. Exercise 9 and the Animated Figures

C. Testing the Phillips Mechanism CH2OH CH2OH

O +
The Phillips mechanism was formulated largely on the ba- H H O
..

..

H + H
sis of structural investigations of lysozyme and a knowl-
.

O C H O C H
edge of the mechanism of nonenzymatic acetal hydrolysis. OR H OR H
A variety of evidence has since been gathered that bears
H NHCOCH3 H NHCOCH3
on the validity of this mechanism. In the remainder of this
section, we discuss the highlights of these studies to illus-
trate how scientific models evolve. CH3

R = H (NAG) or CH (NAM)
a. Identification of the Catalytic Residues
_
Lysozyme’s catalytically important groups have been COO
experimentally identified through site-directed mutagene-
FIGURE 15-15 The D-ring oxonium ion intermediate in the
sis (Section 5-5G) and the use of group-specific reagents:
Phillips mechanism is stabilized by resonance. This requires
Glu 35. The mutagenesis of Glu 35 to Gln yields a protein that atoms C1, C2, C5, and O5 be coplanar (shading); that is,
with no detectable catalytic activity ( 0.1% of wild type), the hexose ring must assume the half-chair conformation.
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Section 15–2. Lysozyme 513

although it has only a
1.5-fold decrease in substrate affin- CH2OH CH2OH

O +
ity. Glu 35 must therefore be essential for lysozyme’s H H O
H H –
catalytic activity. O O
OH H OH H
Asp 52. The mutagenesis of Asp 52 to Asn, which has a O O
polarity comparable to that of Asp but lacks its negative H NHCOCH3 H NHCOCH3
charge, yields an enzyme with no more than 5% of wild- (NAG)3 (NAG)3
type lysozyme’s catalytic activity even though this mutation FIGURE 15-16 The -lactone analog of (NAG)4. Its C1, O1,
causes an
2-fold increase in the enzyme’s affinity for C2, C5, and O5 atoms are coplanar (shading) because of
substrate. Asp 52 is therefore important for enzymatic resonance, as is the D-ring in the reaction intermediate of the
activity. Phillips mechanism (compare with Fig. 15-15).
Noninvolvement of Other Amino Acid Residues.
Lysozyme’s other carboxyl groups besides Glu 35 and Asp
52 do not participate in the catalytic process, as was
demonstrated by reacting lysozyme with carboxyl-specific since this compound’s lactone ring has the half-chair con-
reagents in the presence of substrate. This treatment yields formation that geometrically resembles the proposed oxo-
an almost fully active enzyme in which all carboxyl groups nium ion transition state of the substrate’s D-ring. X-Ray
but Glu 35 and Asp 52 are derivatized. Other group-specific studies indicate, in accordance with prediction, that this in-
reagents that modify, for instance, His, Lys, Met, or Tyr hibitor binds to lysozyme’s A¬ B¬ C ¬ D subsites such
residues but induce no major protein structure disruptions that the lactone ring occupies the D subsite in a half-
cause little change in lysozyme’s catalytic efficiency. chairlike conformation.
Despite the foregoing, the role of substrate distortion in
b. Role of Strain lysozyme catalysis had been questioned. Theoretical stud-
Many of the mechanistic investigations of lysozyme have ies by Michael Levitt and Arieh Warshel on substrate bind-
had the elusive goal of establishing the catalytic role of ing by lysozyme suggested that the protein is too flexible
strain. Not all of these studies, as we shall see, have sup- to mechanically distort the D-ring of a bound substrate.
ported the Phillips mechanism, thereby stimulating a se- Rather, these calculations implied that transition state sta-
ries of investigations that have only recently settled this bilization occurs through the displacement by substrate of
issue. several tightly bound water molecules from the D subsite.
Measurements of the binding equilibria of various The resulting desolvation of the Asp 52 carboxylate group
oligosaccharides to lysozyme indicate that all saccharide would significantly enhance its capacity to electrostatically
residues except that binding to the D subsite contribute en- stabilize the transition state oxonium ion. This study there-
ergetically toward the binding of substrate to lysozyme; fore concluded that “electrostatic strain” rather than steric
binding NAM in the D subsite requires a free energy input strain is the more important factor in stabilizing lysozyme’s
of 12 kJ  mol1 (Table 15-3). The Phillips mechanism transition state.
explains this observation as being indicative of the energy In an effort to obtain further experimental information
penalty of straining the D-ring from its preferred chair bearing on the Phillips strain mechanism, Nathan Sharon
conformation toward the half-chair form. and David Chipman determined the D subsite–binding
As we have discussed in Section 15-1F, an enzyme that affinities of several saccharides by comparing the
catalyzes a reaction by the preferential binding of its tran- lysozyme-binding affinities of various substrate analogs.
sition state has a greater binding affinity for an inhibitor The NAG lactone inhibitor binds to the D subsite with 9.2
that has the transition state geometry (transition state ana- kJ  mol1 greater affinity than does NAG. This quantity
log) than it does for its substrate. The -lactone analog of corresponds, according to Eq. [14.15], to no more than an
(NAG)4 (Fig. 15-16) is a transition state analog of lysozyme
40-fold rate enhancement of the lysozyme reaction as a
result of strain (recall that the difference in binding energy
between a transition state analog and a substrate is in-
dicative of the enzyme’s rate enhancement arising from the
TABLE 15-3 Binding Free Energies of HEW Lysozyme
preferential binding of the transition state complex). Such
Subsites
an enhancement is hardly a major portion of lysozyme’s
Binding
108-fold rate enhancement (accounting for only
20%
Bound Free Energy of the reaction’s ¢ ¢G‡cat ; Section 14-1C). Moreover, an
Site Saccharide (kJ  mol1) N-acetylxylosamine (XylNAc) residue,
A NAG 7.5
H
B NAM 12.3
H O OH
C NAG 23.8 H
O
D NAM 12.1 OH H
H
E NAG 7.1
F NAM 7.1 H NHCOCH3

Source: Chipman, D.M. and Sharon, N., Science 165, 459 (1969). N -Acetylxylosamine residue
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514 Chapter 15. Enzymatic Catalysis

which lacks the sterically hindered ¬ C6H2OH group of C¬ O single bond). Indeed, no such covalent bond had
NAM and NAG, has only marginally greater binding affin- been observed in any of the numerous X-ray structures
ity for the D subsite (3.8 kJ  mol1) than does NAG containing hen egg white (HEW) lysozyme.
(2.5 kJ  mol1). Yet recall that the Phillips mechanism Despite the foregoing, all other -glycosidases of known
postulates that it is the unfavorable contacts made by this structure that cleave glycosidic linkages with net retention
¬ C6H 2OH group that promotes D-ring distortion. of configuration at the anomeric carbon (as does HEW
Nevertheless, lysozyme does not hydrolyze saccharides lysozyme) have been shown to do so via a covalent
with XylNAc in the D subsite. glycosyl–enzyme intermediate. The active sites of these so-
The apparent inconsistencies among the foregoing ex- called retaining -glycosidases structurally resemble that
perimental observations were largely rationalized by of HEW lysozyme. Moreover, there is no direct evidence
Michael James’ highly accurate (1.5-Å resolution) X-ray indicative of the existence of a long-lived oxonium ion at
crystal structure determination of lysozyme in complex the active site of any retaining -glycosidase, including
with NAM–NAG–NAM. This trisaccharide binds, as ex- HEW lysozyme (the lifetime of a glucosyl oxonium ion in
pected, to the B, C, and D subsites of lysozyme. The NAM water is
1012 s, a time only slightly longer than that of
in the D subsite, in agreement with the Phillips mechanism, a bond vibration). Consequently, there had been a growing
is distorted to the half-chair conformation with its suspicion that the HEW lysozyme reaction also proceeds
¬ C6H2OH group in a nearly axial position due to steric via a covalent intermediate, one between the D-ring’s
clashes that would otherwise occur with the acetamido anomeric carbon (C1) and the side chain carboxyl group
group of the C subsite NAG (although, contrary to the orig- of Asp 52 to form an ester linkage:
inal Phillips mechanism, Glu 35 and Trp 108 are too far
away from the ¬ C6H2OH group to contribute to this dis-
tortion). This strained conformation is stabilized by a CH2OH O
strong hydrogen bond between the D-ring O6 and the H O O C CH2 Asp 52
backbone NH of Val 109 (transition state stabilization). H
Indeed, the mutation of Val 109 to Pro, which lacks the OR H
O H
NH group to make such a hydrogen bond, inactivates the
enzyme. Lysozyme’s lack of hydrolytic activity when H NHCOCH3
XylNAc occupies its D subsite is likewise explained by the
absence of this hydrogen bond and the consequent lesser
stability of the XylNAc ring’s half-chair transition state. This intermediate presumably reacts with H2O in what is
The unexpectedly small free energy differences in bind- essentially the reverse of the reaction leading to its for-
ing NAG, NAG lactone, and XylNAc to the D subsite are mation, thereby yielding the reaction’s second product
explained by the observation that undistorted NAG and (a double-displacement mechanism). In this mechanism,
XylNAc can be modeled into the D subsite as it occurs in the oxonium ion is proposed to be the transition state on
the X-ray structure of the lysozyme NAM–NAG–NAM the way to forming the covalent intermediate, rather than
complex. NAM’s bulky lactyl side chain prevents it from being an intermediate itself.
binding to the D subsite in this manner. If, in fact, HEW lysozyme follows this mechanism, the
reason that its covalent intermediate had never been ob-
c. The Lysoyme Reaction Proceeds via a served is that its rate of breakdown must be much faster
Covalent Intermediate than its rate of formation. Hence, if this intermediate is to
Alternatives to the Phillips mechanism postulate that be experimentally observed, its rate of formation must be
either (1) the carboxyl group of Asp 52 displaces the leav- made significantly greater than its rate of breakdown. To
ing group to form a covalent bond to C1, thereby yielding do so, Stephen Withers capitalized on three phenomena.
a covalent glycosyl–enzyme ester intermediate that is sub- First, if, as postulated, the reaction goes through an oxo-
sequently displaced by water to yield product (a double- nium ion transition state, all steps involving its formation
displacement mechanism); or (2) water directly displaces should be slowed by the electron withdrawing effects of
the leaving group (a single-displacement mechanism). A substituting F (the most electronegative element) at C2 of
single-displacement mechanism would result in inversion the D-ring. Second, mutating Glu 35 to Gln (E35Q) re-
of configuration between substrate and product and thus moves the general acid–base that catalyzes the reaction,
can be ruled out. A double-displacement mechanism further slowing all steps involving the oxonium ion transi-
would account for the observed retention of configuration tion state. Third, substituting an additional F atom at C1
in the lysozyme reaction (as does the Phillips mechanism). of the D-ring accelerates the formation of the intermedi-
However, it is at odds with the observation that the dis- ate because this F is a good leaving group. Making all three
tance between C1 in a D subsite–bound saccharide and a of these changes should increase the rate of formation
carboxyl O of Asp 52 (which participates in a network of of the proposed covalent intermediate relative to its
hydrogen bonds that apparently hold this side chain in its breakdown and hence should result in its accumulation.
position) are too long to form a covalent bond (minimally Withers therefore incubated E35Q HEW lysozyme with
2.3 Å in the NAM–NAG–NAM complex without signifi- NAG-(1S 4)-2-deoxy-2-fluoro--D-glucopyranosyl fluo-
cantly disrupting the protein structure vs
1.4 Å for a ride (NAG2FGlcF):
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Section 15–3. Serine Proteases 515

CH2OH CH2OH torted chair conformation, thus indicating that it is a reac-

H O H O F tion intermediate rather than an approximation of the tran-
H H sition state. The superposition of this covalent complex with
OH H O OH H that of the above described complex of NAM–NAG–NAM
HO H H
with wild-type HEW lysozyme reveals how this covalent
H NHCOCH3 H F bond forms (Fig. 15-17). The shortening of the 3.2-Å dis-
NAG2FGlcF tance between the D-ring NAG C1 and the Asp 52 O in
Electrospray ionization mass spectrometry (ESI-MS; Sec- the NAM–NAG–NAM complex to
1.4 Å in the covalent
tion 7-1J) of this reaction mixture revealed a sharp peak complex is almost entirely a consequence of the relaxation
at 14,683 D, consistent with the formation of the proposed of the D-ring from the half-chair to the chair conformation
covalent intermediate, but no significant peak at or near combined with an
45 rotation of the Asp 52 side chain
the 14,314-D molecular mass of the mutant enzyme alone. about its C ¬ C bond; the positions of the D-ring O4 and
The X-ray structure of this covalent complex unam- O6 atoms are essentially unchanged. Hence, over 35 years
biguously reveals the expected
1.4-Å-long covalent bond after its formulation, it was shown that the Phillips mecha-
between C1 of the D-ring NAG and a side chain carboxyl nism must be altered to take into account the transient for-
O of Asp 52 (Fig. 15-17). This D-ring NAG adopts an undis- mation of this covalent glycosyl–enzyme ester intermediate
(covalent catalysis). Keep in mind, however, that in order
to form this covalent linkage, the D-ring must pass through
an oxonium-like transition state, which requires it to tran-
siently assume the half-chair conformation.

3
SERINE PROTEASES
Our next example of enzymatic mechanisms is a diverse
group of proteolytic enzymes known as the serine proteases
(Table 15-4). These enzymes are so named because they
have a common catalytic mechanism characterized by the
possession of a peculiarly reactive Ser residue that is essen-
tial for their enzymatic activity. The serine proteases are the
most thoroughly understood family of enzymes as a result
of their extensive examination over a nearly 50-year period
by kinetic, chemical, physical, and genetic techniques. In this
section, we mainly study the best characterized serine pro-
teases, chymotrypsin, trypsin, and elastase. We also consider
how these three enzymes, which are synthesized in inactive
forms, are physiologically activated.

A. Kinetics and Catalytic Groups

Chymotrypsin, trypsin, and elastase are digestive enzymes
that are synthesized by the pancreatic acinar cells (Fig. 1-10c)
and secreted, via the pancreatic duct, into the duodenum (the
small intestine’s upper loop). All of these enzymes catalyze
FIGURE 15-17 The HEW lysozyme covalent intermediate. The the hydrolysis of peptide (amide) bonds but with different
substrate C- and D-rings and Asp 52 are shown in the specificities for the side chains flanking the scissile (to be
superposition of the X-ray structures of the covalent complex cleaved) peptide bond (recall that chymotrypsin is specific
formed by reacting E35Q lysozyme with NAG2FGlcF (C green, for a bulky hydrophobic residue preceding the scissile pep-
N blue, O red, and F magenta) and the noncovalent complex of tide bond, trypsin is specific for a positively charged residue,
wild-type lysozyme with NAM–NAG–NAM (C yellow, N blue, and elastase is specific for a small neutral residue; Table 7-
and O red). Note that the covalent bond between Asp 52 and 2). Together, they form a potent digestive team.
C1 of the D-ring forms when the D-ring in the noncovalent
complex relaxes from its distorted half-chair conformation to an
a. Ester Hydrolysis as a Kinetic Model
undistorted chair conformation and that the side chain of Asp
52 undergoes an
45 rotation about its C¬ C bond. [Based
That chymotrypsin can act as an esterase as well as a
on X-ray structures by David Vocadlo and Stephen Withers, protease is not particularly surprising since the chemical
University of British Columbia, Vancouver, Canada; and mechanisms of ester and amide hydrolysis are almost iden-
Michael James, University of Alberta, Edmonton, Canada. tical. The study of chymotrypsin’s esterase activity has led
PDBids 1H6M and 9LYZ.] to important insights concerning this enzyme’s catalytic
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516 Chapter 15. Enzymatic Catalysis

TABLE 15-4 A Selection of Serine Proteases

Enzyme Source Function
Trypsin Pancreas Digestion of proteins
Chymotrypsin Pancreas Digestion of proteins
Elastase Pancreas Digestion of proteins
Thrombin Vertebrate serum Blood clotting
Plasmin Vertebrate serum Dissolution of blood clots
Kallikrein Blood and tissues Control of blood flow
Complement C1 Serum Cell lysis in the immune response
Acrosomal protease Sperm acrosome Penetration of ovum
Lysosomal protease Animal cells Cell protein turnover
Cocoonase Moth larvae Dissolution of cocoon after metamorphosis
-Lytic protease Bacillus sorangium Possibly digestion
Proteases A and B Streptomyces griseus Possibly digestion
Subtilisin Bacillus subtilis Possibly digestion
Source: Stroud, R.M., Sci. Am. 231(1), 86 (1974).

FIGURE 15-18 Time course of p-nitrophenylacetate hydrolysis 4

as catalyzed by two different concentrations of chymotrypsin. Burst Steady state
The enzyme rapidly binds substrate and releases the first phase phase L–1
g⋅m
0.8 m
[ p-Nitrophenolate] (mM)

product, p-nitrophenolate ion, but the second product, acetate 3

ion, is released more slowly. Consequently, the rate of
p-nitrophenolate generation begins rapidly (burst phase) but
slows as acyl–enzyme complex accumulates until the rate of
2
p-nitrophenolate generation approaches that of acetate release –1
(steady state). The extrapolation of the steady state curve to 0.4 mg ⋅ mL
zero time (dashed lines) indicates the initial concentration of
active enzyme. [After Hartley, B.S. and Kilby, B.A., Biochem. J. 1
56, 294 (1954).]

0
2 4 6 8 10 12
mechanism. Kinetic measurements by Brian Hartley of the Time (min)
chymotrypsin-catalyzed hydrolysis of p-nitrophenylacetate
O
p-nitrophenolate ion forming a covalent acyl–enzyme in-
CH3 C O NO2 termediate that (2) is slowly hydrolyzed to release acetate:
O
p-Nitrophenylacetate
CH3 C O NO2 + Enzyme
H2O
chymotrypsin
2H+ p-Nitrophenylacetate Chymotrypsin
O
fast –O NO2
CH3 C O– + –O NO2

p-Nitrophenolate
Acetate p-Nitrophenolate
indicated that the reaction occurs in two phases (Fig. 15-18): O

1. The “burst phase,” in which the highly colored CH3 C Enzyme

p-nitrophenolate ion is rapidly formed in amounts Acyl–enzyme intermediate
stoichiometric with the quantity of active enzyme present.
H2O
2. The “steady-state phase,” in which p-nitrophenolate slow
is generated at a reduced but constant rate that is inde- H+
pendent of substrate concentration.
O
These observations have been interpreted in terms of a
two-stage reaction sequence in which the enzyme (1) CH3 C O– + Enzyme
rapidly reacts with the p-nitrophenylacetate to release Acetate
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Section 15–3. Serine Proteases 517

Chymotrypsin evidently follows a Ping Pong Bi Bi mech- types of nerve cells (Sections 12-4D and 20-5C). The in-
anism (Section 14-5A). Chymotrypsin-catalyzed amide activation of acetylcholinesterase prevents the otherwise
hydrolysis has been shown to follow a reaction pathway rapid hydrolysis of the acetylcholine released by a nerve
similar to that of ester hydrolysis but with the first step of impulse and thereby interferes with the regular sequence
the reaction, enzyme acylation, being rate determining of nerve impulses. DIPF is of such great toxicity to humans
rather than the deacylation step. that it has been used militarily as a nerve gas. Related com-
pounds, such as parathion and malathion,
b. Identification of the Catalytic Residues O CH2CH3
Chymotrypsin’s catalytically important groups were
O 2N O P S
identified by chemical labeling studies. These are described
below. O CH2CH3

Ser 195. A diagnostic test for the presence of the active Parathion
Ser of serine proteases is its reaction with diisopropyl-
phosphofluoridate (DIPF): O
CH3 CH2 O C O CH3
CH(CH3)2
O CH S P S
O
CH3 CH2 O C CH2 O CH3
(Active Ser) CH2OH + F P O
Malathion
O
are useful insecticides because they are far more toxic to
CH(CH3)2 insects than to mammals.
Diisopropylphospho- His 57. A second catalytically important residue was dis-
fluoridate (DIPF) covered through affinity labeling. In this technique, a sub-
strate analog bearing a reactive group specifically binds at
the enzyme’s active site, where it reacts to form a stable
CH(CH3)2 covalent bond with a nearby susceptible group (these
reactive substrate analogs have therefore been described
O as the “Trojan horses” of biochemistry). The affinity la-
(Active Ser) CH2 O P O + HF
beled groups can subsequently be identified by peptide
mapping (Section 7-1K). Chymotrypsin specifically binds
O tosyl-L-phenylalanine chloromethyl ketone (TPCK),
CH(CH3)2

DIP–Enzyme

which irreversibly inactivates the enzyme. Other Ser O CH2 O

residues, including those on the same protein, do not react
with DIPF. DIPF reacts only with Ser 195 of chymotrypsin, CH3 S NH CH C CH2Cl
thereby demonstrating that this residue is the enzyme’s active O
Ser. because of its resemblance to a Phe residue (one of chy-
The use of DIPF as an enzyme inactivating agent came motrypsin’s preferred residues; Table 7-2). Active site–
about through the discovery that organophosphorus com- bound TPCK’s chloromethyl ketone group is a strong
pounds such as DIPF are potent nerve poisons. The neu- alkylating agent; it reacts with His 57 (Fig. 15-19), thereby
rotoxicity of DIPF arises from its ability to inactivate
acetylcholinesterase, a serine esterase that catalyzes the
hydrolysis of acetylcholine:
O Chymotrypsin Chymotrypsin
+
(CH3)3N CH2 CH2 O C CH3 + H2O CH2 H
Cl CH2
HCl
Acetylcholine N N
CH2
acetylcholinesterase
N + C O N
O
O
R CH2
+
(CH3)3N CH2 CH2 OH + C CH3
TPCK C O
His 57
Choline –O
R
Acetylcholine is a neurotransmitter: It transmits nerve im- FIGURE 15-19 Reaction of TPCK with chymotrypsin to
pulses across the synapses (junctions) between certain alkylate His 57.
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518 Chapter 15. Enzymatic Catalysis

(a)
(a)

FIGURE 15-20 X-Ray structure of bovine trypsin. (a) A Reproduced with permission.] (b) A ribbon diagram of trypsin
drawing of the enzyme in complex with a polypeptide substrate highlighting its secondary structure and indicating the
(green) that has its Arg side chain occupying the enzyme’s arrangement of its catalytic triad. (c) A drawing showing the
specificity pocket (stippling). The C backbone of the enzyme is surface of trypsin (blue) superimposed on its polypeptide
shown together with its disulfide bonds and the side chains of backbone ( purple). The side chains of the catalytic triad are
the catalytic triad, Ser 195, His 57, and Asp 102. The active shown in green. [Courtesy of Arthur Olson, The Scripps
sites of chymotrypsin and elastase contain almost identically Research Institute, La Jolla, California.] Parts a, b, and c have
arranged catalytic triads. [Illustration, Irving Geis/Geis approximately the same orientation. See Kinemage
Archives Trust. Copyright Howard Hughes Medical Institute. Exercise 10-1
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Section 15–3. Serine Proteases 519

inactivating the enzyme. The TPCK reaction is inhibited amino acid residue numbering scheme. Bovine chymo-
by -phenylpropionate, trypsin is synthesized as an inactive 245-residue precursor
named chymotrypsinogen that is proteolytically converted
to chymotrypsin (Section 15-3E). In what follows, the num-
bering of the amino acid residues in chymotrypsin, trypsin,
and elastase will be that of the corresponding residues in
bovine chymotrypsinogen.
CH2 CH2 COO–
The X-ray structure of bovine chymotrypsin was eluci-
-Phenylpropionate dated in 1967 by David Blow. This was followed by the de-
termination of the structures of bovine trypsin (Fig. 15-20)
a competitive inhibitor of chymotrypsin that presumably by Robert Stroud and Richard Dickerson, and porcine
competes with TPCK for its enzymatic binding site. elastase by David Shotton and Herman Watson. Each of
Moreover, the TPCK reaction does not occur in 8M urea, these proteins is folded into two domains, both of which
a denaturing reagent, or with DIP–chymotrypsin, in which have extensive regions of antiparallel -sheets in a barrel-
the active site is blocked. These observations establish that like arrangement but contain little helix. The catalytically
His 57 is an essential active site residue of chymotrypsin. essential His 57 and Ser 195 are located at the substrate-
binding site together with the invariant (in all serine pro-
B. X-Ray Structures teases) Asp 102, which is buried in a solvent-inaccessible
pocket. These three residues form a hydrogen bonded con-
Bovine chymotrypsin, bovine trypsin, and porcine elastase
stellation referred to as the catalytic triad (Figs. 15-20 and
are strikingly homologous: The primary structures of these
15-21).

240-residue monomeric enzymes are
40% identical and
their internal sequences are even more alike (in compari-
a. The Structural Basis of Substrate Specificity Can Be
son, the  and  chains of human hemoglobin have a 44%
Quite Complex
sequence identity). Furthermore, all of these enzymes have
The X-ray structures of the above three enzymes sug-
an active Ser and a catalytically essential His as well as sim-
gest the basis for their differing substrate specificities
ilar kinetic mechanisms. It therefore came as no surprise
(Table 7-2):
when their X-ray structures all proved to be closely related.
To most conveniently compare the structures of these 1. In chymotrypsin, the bulky aromatic side chain of
three digestive enzymes, they have been assigned the same the preferred Phe, Trp, or Tyr residue that contributes the

His 57
Ser 195 N

Asp 102

(b) (c)
FIGURE 15-20 (Continued)
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520 Chapter 15. Enzymatic Catalysis

Ser 195 Gly 193

Catalytic
triad

Asp 194

His 57
Ile 16

FIGURE 15-21 The active site residues of chymotrypsin. The

view is in approximately the same direction as in Fig. 15-20.
Asp 102 The catalytic triad consists of Ser 195, His 57, and Asp 102.
[After Blow, D.M. and Steitz, T.A., Annu. Rev. Biochem. 39, 86
(1970).]

carbonyl group of the scissile peptide fits snugly into a slit- a poor, nonspecific protease. Moreover, even replacing the
like hydrophobic pocket, the specificity pocket, that is lo- other three residues in trypsin’s specificity pocket that
cated near the catalytic groups (Fig. 15-20a). differ from those in chymotrypsin, with those of chymo-
2. In trypsin, the residue corresponding to chymo- trypsin, fails to yield a significantly improved enzyme.
trypsin Ser 189, which lies at the back of the specificity However, trypsin is converted to a reasonably active
pocket, is the anionic residue Asp. The cationic side chains chymotrypsin-like enzyme when, in addition to the fore-
of trypsin’s preferred residues, Arg or Lys, can therefore going changes (collectively designated S1), both of its
form ion pairs with this Asp residue. The rest of chymo- two surface loops that connect the walls of the speci-
trypsin’s specificity pocket is preserved in trypsin so that it ficity pocket, L1 (residues 185–188) and L2 (residues
can accommodate the bulky side chains of Arg and Lys. 221–225), are replaced by those of chymotrypsin (termed
TrSCh[S1 L1 L2]). Although this mutant enzyme still
3. Elastase is so named because it rapidly hydrolyzes has a low substrate-binding affinity, KS, the additional mu-
the otherwise nearly indigestible Ala, Gly, and Val-rich tation Y172W in a third surface loop yields an enzyme
protein elastin (a connective tissue protein with rubberlike (TrSCh[S1 L1 L2+Y172W]) that has 15% of chymo-
elastic properties). Elastase’s specificity pocket is largely trypsin’s catalytic efficiency. Curiously, these loops, whose
occluded by the side chains of a Val and a Thr residue that sequences are largely conserved in each enzyme, are not
replace two Gly’s lining this pocket in both chymotrypsin structural components of either the specificity pocket or
and trypsin. Consequently elastase, whose specificity the extended substrate binding site in chymotrypsin or in
pocket is better described as a depression, specifically trypsin (Fig. 15-20a).
cleaves peptide bonds after small neutral residues, partic- Careful comparisons, by Charles Craik and Robert
ularly Ala. In contrast, chymotrypsin and trypsin hydrolyze Fletterick, of the X-ray structures of chymotrypsin and
such peptide bonds extremely slowly because these small trypsin with those of the closely similar TrSCh[S1 L1
substrates cannot be sufficiently immobilized on the en- L2] and TrSCh[S1 L1 L2 Y172W] in complex with a
zyme surface for efficient catalysis to occur (Section Phe-containing chloromethyl ketone inhibitor reveal the
15-1E). structural basis of substrate specificity in trypsin and
Thus, for example, trypsin catalyzes the hydrolysis of pepti- chymotrypsin. Efficient catalysis in the serine proteases re-
dyl amide substrates with an Arg or Lys residue preceding quires that the enzyme’s active site be structurally intact
the scissile bond with an efficiency, as measured by kcatKM and that the substrate’s scissile bond be properly positioned
(Section 14-2B), that is 106-fold greater than that for relative to the catalytic triad and other components of the
the corresponding Phe-containing substrates. Conversely, active site (see below). The above mutagenic changes do
chymotrypsin catalyzes the hydrolysis of substrates after not affect the structure of the catalytic triad or those por-
Phe, Trp, and Tyr residues 104-fold more efficiently than tions of the active site that bind the substrate’s leaving
after the corresponding Lys-containing substrates. group (that segment on the C-terminal side of the scissile
Despite the foregoing, the mutagenic change in trypsin bond). However, the main chain conformation of the con-
of Asp 189 S Ser (D189S) by William Rutter did not switch served Gly 216 (which forms two hydrogen bonds to the
its specificity to that of chymotrypsin but instead yielded backbone of the third residue before the substrate’s scis-
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Section 15–3. Serine Proteases 521

sile bond in an antiparallel
pleated sheet–like arrange- Serine ClpP

Subtilisin Chymotrypsin carboxypeptidase II protease
ment) differs in trypsin and chymotrypsin and adopts a
NH+3 NH+3 NH+3 NH+3
chymotrypsin-like structure in both hybrid proteins.
Evidently, if Gly 216 adopts a trypsin-like conformation, Asp 32
the scissile bond in Phe-containing substrates is misori-
ented for efficient catalysis. Thus, despite the fact that Gly His 64
216 is conserved in trypsin and chymotrypsin, the differing Ser
His 57 146
structures of loop L2 in the two enzymes maintain it in dis-
tinct conformations. Asp
Loop L1, which interacts with L2 in both trypsin and Ser 125 102
chymotrypsin, is largely disordered in the X-ray structure Leu 126
Gly 127 Ser
of TrSCh[S1L1L2]. Modeling a trypsin-like L1 into 97
TrSCh[S1L1L2] results in severe steric clashes with
the chymotrypsin-like L2. Thus, the requirement of a chy- Asp
338 His
motrypsin-like L1 for the efficient catalysis by TrS 122
Ch[S1L1L2] appears to arise from the need to permit Ser 195
L2 to adopt a chymotrypsin-like conformation. His
Ser 221 Ser 214 397 Asp
Residue 172 is located at the base of the specificity Trp 215 171
pocket. The improvement in substrate binding affinity of Gly 216
TrSCh[S1L1L2Y172W] over TrSCh[S1L1L2]
arises from structural rearrangements in this region of the
enzyme caused by the increased bulk and different COO– COO– COO– COO–
hydrogen bonding requirements of Trp versus Tyr. These FIGURE 15-22 Relative positions of the active site residues in
changes appear to improve both the structural stability of subtilisin, chymotrypsin, serine carboxypeptidase II, and ClpP
residues forming the specificity pocket and their specificity protease. The peptide backbones of Ser 214, Trp 215, and Gly
for chymotrypsin-like substrates. These results therefore 216 in chymotrypsin, and their counterparts in subtilisin,
highlight an important caveat for genetic engineers: participate in substrate-binding interactions. [After Robertus,
Enzymes are so exquisitely tailored to their functions that J.D., Alden, R.A., Birktoft, J.J., Kraut, J., Powers, J.C., and
Wilcox, P.E., Biochemistry 11, 2449 (1972).] See Kinemage
they often respond to mutagenic tinkering in unexpected
Exercise 10-2
ways.

b. Evolutionary Relationships among Serine Proteases

carboxypeptidase A (Fig. 8-19a) even though the latter
We have seen that sequence and structural homologies
protease has an entirely different catalytic mechanism from
among proteins reveal their evolutionary relationships
that of the serine proteases (see Problem 3).
(Sections 7-3 and 9-6). The great similarities among chy-
motrypsin, trypsin, and elastase indicate that these proteins 3. E. coli ClpP, which functions in the degradation of
evolved through gene duplications of an ancestral serine cellular proteins (Section 32-6B).
protease followed by the divergent evolution of the result- Since the orders of the corresponding active site residues
ing enzymes (Section 7-3C). in the amino acid sequences of the four types of serine
Several serine proteases from various sources provide proteases are quite different (Fig. 15-22), it seems highly
further insights into the evolutionary relationships among improbable that they could have evolved from a common
the serine proteases. Streptomyces griseus protease A ancestor serine protease. These proteins apparently consti-
(SGPA) is a bacterial serine protease of chymotryptic tute a remarkable example of convergent evolution: Nature
specificity that exhibits extensive structural similarity, al- seems to have independently discovered the same catalytic
though only
20% sequence identity, with the pancreatic mechanism at least four times. (In addition, human cy-
serine proteases. The primordial trypsin gene evidently tomegalovirus protease, an essential protein for virus repli-
arose before the divergence of prokaryotes and eukary- cation that bears no resemblance to the above proteases,
otes. has active site Ser and His residues whose relative posi-
There are three known serine proteases whose primary tions are similar to those in other serine proteases but lacks
and tertiary structures bear no discernible relationship to an active site Asp residue; it appears to have a catalytic
each other or to chymotrypsin but which, nevertheless, dyad.)
contain catalytic triads at their active sites whose structures
closely resemble that of chymotrypsin:
C. Catalytic Mechanism
1. Subtilisin, an endopeptidase that was originally iso- See Guided Exploration 12: The Catalytic Mechanism of Serine
lated from Bacillus subtilis. Proteases The extensive active site homologies among the
2. Wheat germ serine carboxypeptidase II, an exopep- various serine proteases indicate that they all have the
tidase whose structure is surprisingly similar to that of same catalytic mechanism. On the basis of considerable
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522 Chapter 15. Enzymatic Catalysis

chemical and structural data gathered in many laborato- 1. After chymotrypsin has bound substrate to form the
ries, the following catalytic mechanism has been formu- Michaelis complex, Ser 195, in the reaction’s rate-deter-
lated for the serine proteases, here given in terms of chy- mining step, nucleophilically attacks the scissile peptide’s
motrypsin (Fig. 15-23): carbonyl group to form a complex known as the tetrahedral

Asp His Asp His

102 57 102 57
CH2 CH2
.O .O
H 2C ... H 2C ...
C – H Ser C – H Ser

....
....

N1 195 N1 195
O O
3 3
CH2 N+ CH2
..

N
1
H O H O
Nucleophilic
R R
attack
R R N C
Substrate
polypeptide
N C H O–
H O Tetrahedral intermediate

Enzyme–substrate
complex 2

Asp His
Asp His 57
102
102 57
CH2
.O
CH2 H 2C ...
.O C – H Ser
H2C ...
C –
....
H Ser N1 195
H2O O
....

N1 195 3
O 2
3 N CH2
N CH2
O
New N-terminus of H
O RNH2 R R
cleaved polypeptide
H R chain N C
O C
H O
H O
Acyl–enzyme intermediate

Asp His
Asp His 57
102
102 57
CH2
.O
O CH2 H 2C ...
H2C . ... C – H Ser
C – H
....

Ser N 195
O
....

N 195
O 4 N CH2
N+ CH2
H O
H O
R
+
R
New C-terminus
O C of cleaved polypeptide O C
H O– chain
H O
Tetrahedral intermediate Active enzyme

FIGURE 15-23 Catalytic mechanism of the serine proteases. active site Asp-polarized His, followed by loss of the amine
The reaction involves (1) the nucleophilic attack of the active product and its replacement by a water molecule; (3) the
site Ser on the carbonyl carbon atom of the scissile peptide reversal of Step 2 to form a second tetrahedral intermediate; and
bond to form the tetrahedral intermediate; (2) the (4) the reversal of Step 1 to yield the reaction’s carboxyl
decomposition of the tetrahedral intermediate to the acyl– product and the active enzyme.
enzyme intermediate through general acid catalysis by the
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Section 15–3. Serine Proteases 523

intermediate (covalent catalysis). X-Ray studies indicate The portion of BPTI in contact with the trypsin active
that Ser 195 is ideally positioned to carry out this nucleo- site resembles bound substrate. The side chain of BPTI Lys
philic attack (proximity and orientation effects). The imi- 15I (here “I” differentiates BPTI residues from trypsin
dazole ring of His 57 takes up the liberated proton, thereby residues) occupies the trypsin specificity pocket (Fig.
forming an imidazolium ion (general base catalysis). This 15-24a) and the peptide bond between Lys 15I and Ala 16I
process is aided by the polarizing effect of the unsolvated is positioned as if it were the scissile peptide bond
carboxylate ion of Asp 102, which is hydrogen bonded to (Fig. 15-24b). What is most remarkable about this structure
His 57 (electrostatic catalysis; see Section 15-3D). Indeed, is that its active site complex assumes a conformation well
the mutagenic replacement of trypsin’s Asp 102 by Asn along the reaction coordinate toward the tetrahedral
leaves the enzyme’s KM substantially unchanged at neutral intermediate: The side chain oxygen of trypsin Ser 195, the
pH but reduces its kcat to 0.05% of its wild-type value. active Ser, is in closer-than-van der Waals contact (2.6 Å)
Neutron diffraction studies have demonstrated that Asp with the pyramidally distorted carbonyl carbon of BPTI’s
102 remains a carboxylate ion rather than abstracting a pro- “scissile” peptide. Despite this close contact, the proteolytic
ton from the imidazolium ion to form an uncharged car- reaction cannot proceed past this point along the reaction
boxylic acid group. The tetrahedral intermediate has a coordinate because of the rigidity of the active site com-
well-defined, although transient, existence. We shall see plex and because it is so tightly sealed that the leaving group
that much of chymotrypsin’s catalytic power derives from cannot leave and water cannot enter the reaction site.
its preferential binding of the transition state leading to this
Protease inhibitors are common in nature, where they have
intermediate (transition state binding catalysis).
protective and regulatory functions. For example, certain
2. The tetrahedral intermediate decomposes to the
acyl–enzyme intermediate under the driving force of pro-
ton donation from N3 of His 57 (general acid catalysis).
The amine leaving group (RNH2, the new N-terminal por-
tion of the cleaved polypeptide chain) is released from the
enzyme and replaced by water from the solvent.
3 & 4. The acyl-enzyme intermediate (which, in the ab-
sence of enzyme, would be a stable compound) is rapidly
deacylated by what is essentially the reverse of the previ-
ous steps followed by the release of the resulting carboxy-
late product (the new C-terminal portion of the cleaved
polypeptide chain), thereby regenerating the active en-
zyme. In this process, water is the attacking nucleophile
and Ser 195 is the leaving group.

D. Testing the Catalytic Mechanism

The formulation of the foregoing model for catalysis by (a)
serine proteases has prompted numerous investigations of
its validity. In this section we discuss several of the most Ser 195
H
revealing of these studies. O Ala 16I
O C
a. The Tetrahedral Intermediate Is Mimicked in a C N
Complex of Trypsin with Trypsin Inhibitor
C H
Convincing structural evidence for the existence of the
(b)
(b) Lys 15I
tetrahedral intermediate was provided by Robert Huber
in an X-ray study of the complex between bovine pancre- FIGURE 15-24 Trypsin–BPTI complex. (a) The X-ray
atic trypsin inhibitor (BPTI) and trypsin. The 58-residue structure shown as a cutaway surface drawing indicating how
protein BPTI, whose folding pathway we examined in trypsin (red) binds BPTI (green). The green protrusion
Section 9-1C, binds to and inactivates trypsin, thereby pre- extending into the red cavity near the center of the figure
venting any trypsin that is prematurely activated in the represents the Lys 15I side chain occupying trypsin’s specificity
pocket. Note the close complementary fit of these two proteins.
pancreas from digesting that organ (see Section 15-3E).
[Courtesy of Michael Connolly, New York University.]
BPTI binds to the active site region of trypsin across
(b) Trypsin Ser 195, the active Ser, is in closer-than-van der
a tightly packed interface that is cross-linked by a com- Waals contact with the carbonyl carbon of BPTI’s scissile
plex network of hydrogen bonds. This complex’s 1013M1 peptide, which is pyramidally distorted toward Ser 195. The
association constant, among the largest of any known pro- normal proteolytic reaction is apparently arrested somewhere
tein–protein interaction, emphasizes BPTI’s physiological along the reaction coordinate between the Michaelis complex
importance. and the tetrahedral intermediate.
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524 Chapter 15. Enzymatic Catalysis

plants release protease inhibitors in response to insect premise that convergent evolution had made the active
bites, thereby causing the offending insect to starve by in- sites of these unrelated enzymes functionally identical.
activating its digestive enzymes. Protease inhibitors consti-
tute
10% of the nearly 200 proteins of blood serum. For
3. The tetrahedral distortion, moreover, permits the
instance,
1-proteinase inhibitor, which is secreted by the formation of an otherwise unsatisfied hydrogen bond be-
liver, inhibits leukocyte elastase (leukocytes are a type of tween the enzyme and the backbone NH group of the
white blood cell; the action of leukocyte elastase is thought residue preceding the scissile peptide. Consequently, the
to be part of the inflammatory process). Pathological vari- enzyme binds the tetrahedral intermediate in preference to
ants of 1-proteinase inhibitor with reduced activity are as- either the Michaelis complex or the acyl–enzyme interme-
sociated with pulmonary emphysema, a degenerative dis- diate.
ease of the lungs resulting from the hydrolysis of its elastic
fibers. Smokers also suffer from reduced activity of their It is this phenomenon that is responsible for much of the
1-proteinase inhibitor because of the oxidation of its ac- catalytic efficiency of serine proteases (see below). In fact,
tive site Met residue. Full activity of this inhibitor is not re- the reason that DIPF is such an effective inhibitor of ser-
gained until several hours after smoking. ine proteases is because its tetrahedral phosphate group
makes this compound a transition state analog of the
b. Serine Proteases Preferentially Bind the enzyme.
Transition State
Detailed comparisons of the X-ray structures of several c. The Tetrahedral Intermediate and the Water
serine protease–inhibitor complexes have revealed a fur- Molecule Attacking the Acyl–Enzyme Intermediate
ther structural basis for catalysis in these enzymes (Fig. Have Been Directly Observed
15-25): Most enzymatic reactions turn over far too rapidly for
their intermediate states to be studied by X-ray or NMR
1. The conformational distortion that occurs with the techniques. Consequently, much of our structural knowl-
formation of the tetrahedral intermediate causes the car- edge of these intermediate states derives from the study of
bonyl oxygen of the scissile peptide to move deeper into enzyme–inhibitor complexes or complexes of substrates
the active site so as to occupy a previously unoccupied po- with inactivated enzymes. Yet the structural relevance of
sition, the oxyanion hole. these complexes is subject to doubt precisely because they
2. There it forms two hydrogen bonds with the enzyme are catalytically unproductive.
that cannot form when the carbonyl group is in its normal In an effort to rectify this situation for serine proteases,
trigonal conformation. These two enzymatic hydrogen Janos Hadju and Christopher Schofield searched for
bond donors were first noted by Joseph Kraut to occupy peptide–protease complexes that are stable at pH’s at
corresponding positions in chymotrypsin and subtilisin. He which the protease is inactive but which could be rendered
proposed the existence of the oxyanion hole based on the active by changing the pH. To do so, they screened libraries

(a) (b)
Oxyanion hole

Ser 195 Ser 195

His 57 N His 57
N
H H
...

O
Gly 193 NH O H Gly 193 NH ... O– O
....

C O C O
Asp Asp
.

HN N NH ...–O C 102 R N Cα HN ... H N + N..H..

–O C 102
R N Cα
H
H
...

Cβ Cβ
O R′ O R′

C C
Gly 193 Gly 193

FIGURE 15-25 Transition state stabilization in the serine Gly 193 and Ser 195. The consequent conformational distortion
proteases. (a) In the Michaelis complex, the trigonal carbonyl permits the NH group of the residue preceding the scissile
carbon of the scissile peptide is conformationally constrained peptide bond to form an otherwise unsatisfied hydrogen bond
from binding in the oxyanion hole (upper left). (b) In the to Gly 193. Serine proteases therefore preferentially bind the
tetrahedral intermediate, the now charged carbonyl oxygen of tetrahedral intermediate. [After Robertus, J.D., Kraut, J.,
the scissile peptide (the oxyanion) has entered the oxyanion Alden, R.A., and Birktoft, J.J., Biochemistry 11, 4302 (1972).]
hole, thereby hydrogen bonding to the backbone NH groups of See Kinemage Exercise 10-3
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Section 15–3. Serine Proteases 525

of peptides for their ability to bind to porcine pancreatic nearly perpendicular to the plane of the acyl group). His
elastase at pH 3.5 (at which pH His 57 is protonated and 57, which is hydrogen bonded to this water molecule, is
hence unable to act as a general base) through the use of properly positioned to abstract one of its protons, thereby
ESI-MS (Section 7-1J). They thereby discovered that activating it for the nucleophilic attack (general base cat-
YPFVEPI, a heptapeptide segment of the human milk pro- alysis). The carbonyl O atom of the acyl group occupies
tein -casein that is named BCM7, forms a complex with the enzyme’s oxyanion hole such that it is hydrogen bonded
elastase, whose mass is consistent with the formation of an to the main chain N atoms of both Ser 195 and Gly 193.
ester linkage between BCM7 and the enzyme. In the pres- This is in agreement with spectroscopic measurements in-
ence of 18OH2 at pH 7.5 (where elastase is active), the 18O dicating that the acyl–enzyme intermediate’s carbonyl
label was incorporated into both BCM7 and the elastase– group is, in fact, hydrogen bonded to the oxyanion hole.
BCM7 complex, thereby demonstrating that the reaction It was initially assumed that the oxyanion hole acts only
of BCM7 with elastase is reversible at this pH. to stabilize the tetrahedral oxyanion transition state that
Fragmentation studies by fast atom bombardment–tandem resides near the tetrahedral intermediate on the catalytic
mass spectroscopy (FAB–MS/MS; Section 7-1J) further re- reaction coordinate. However, it now appears that the
vealed that BCM7 that had been incubated with elastase oxyanion hole also functions to polarize the carbonyl group
in the presence of 18OH2 at pH 7.5 incorporated the 18O of the acyl–enzyme intermediate toward an oxyanion
label into only its C-terminal Ile residue. (electrostatic catalysis).
The X-ray structure of the BCM7–elastase complex at The catalytic reaction was initiated in crystals of the
pH 5 (Fig. 15-26a) revealed that BCM7’s C-terminal car- BCM7–elastase complex by transferring them to a buffer
boxyl group, in fact, forms an ester linkage with elastase’s at pH 9. After soaking in this buffer for 1 min, the crystals
Ser 195 side chain hydroxyl group to form the expected were rapidly frozen in liquid N2 (196°C), thereby arrest-
acyl–enzyme intermediate. Moreover, this X-ray structure ing the enzymatic reaction (recall that the catalytically
reveals the presence of a bound water molecule that ap- essential collective motions of proteins cease at such low
pears poised to nucleophilically attack the ester linkage temperatures; Section 9-4). The X-ray structure of such a
(the distance from this water molecule to BCM7’s C- frozen crystal (Fig. 15-26b) revealed that the above acyl–
terminal C atom is 3.1 Å and the line between them is enzyme intermediate had converted to the tetrahedral in-

(a) (b)

FIGURE 15-26 X-Ray structures of porcine pancreatic elastase represent the catalytically important hydrogen bonds and the
in complex with the heptapeptide BCM7 (YPFVEPI). The dotted gray line indicates the trajectory that the bound water
residues of elastase are specified by the three-letter code and molecule presumably follows in nucleophilically attacking the
those of BCM7 are specified by the one-letter code. (a) The acyl group’s carbonyl C atom. (b) The complex after being
complex at pH 5. The enzyme’s active site residues and the brought to pH 9 for 1 min and then rapidly frozen in liquid
heptapeptide (whose N-terminal three residues are disordered) nitrogen. The various groups in the structure are represented
are shown in ball-and-stick form with elastase C green, BCM7 and colored as in Part a. Note that the water molecule in
C cyan, N blue, O red, S yellow, and the bond between the Ser Part a has become a hydroxyl substituent (orange) to the
195 O atom and the C-terminal C atom of BCM7 magenta. The carbonyl C atom, thereby yielding the tetrahedral intermediate.
enzyme-bound water molecule, which appears poised to [Based on X-ray structures by Christopher Schofield and Janos
nucleophilically attack the acyl–enzyme’s carbonyl C atom, is Hadju, University of Oxford, U.K. PDBids (a) 1HAX and (b)
represented by an orange sphere. The dashed gray lines 1HAZ.]
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526 Chapter 15. Enzymatic Catalysis

termediate, whose oxyanion, as expected, remained hy- ference between the pK’s of the donor and acceptor).
drogen bonded to the N atoms of Ser 195 and Gly 193. However, when the pK’s of the hydrogen bonding donor
Comparison of this crystal structure with that of the acyl– (D) and acceptor (A) groups are nearly equal, the dis-
enzyme intermediate reveals that the enzyme’s active site tinction between them breaks down: The hydrogen atom
residues do not significantly change their positions in the becomes more or less equally shared between them
conversion from the acyl–enzyme intermediate to the (D p H p A). Such low-barrier hydrogen bonds (LBHBs)
tetrahedral intermediate. However, the peptide substrate are unusually short and strong (they are also known as
must do so out of steric necessity when the trigonal planar short, strong hydrogen bonds): They have, as studies of
acyl group converts to the tetrahedral oxyanion (compare model compounds in the gas phase indicate, association
Figs. 15-26a and 15-26b). In response, several enzyme free energies as high as 40 to 80 kJ  mol1 versus the
residues that contact the peptide but which are distant from 12 to 30 kJ  mol1 for normal hydrogen bonds (the en-
the active site also shift their positions (not shown in Fig. ergy of the normally covalent D¬ H bond is subsumed
15-26). into the low-barrier hydrogen bonding system) and a
D p A length of 2.55 Å for O¬ H p O and 2.65 Å for
d. The Role of the Catalytic Triad: Low-Barrier N¬ H p O versus 2.8 to 3.1 Å for normal hydrogen bonds.
Hydrogen Bonds LBHBs are unlikely to exist in dilute aqueous solution
The earlier literature postulated that the Asp 102- because water molecules, which are excellent hydrogen
polarized His 57 side chain directly abstracts a proton bonding donors and acceptors, effectively compete with
from Ser 195, thereby converting its weakly nucleophilic D¬ H and A for hydrogen bonding sites. However,
¬ CH2OH group to a highly nucleophilic alkoxide ion, LBHBs may exist in nonaqueous solution and in the active
¬ CH2O: sites of enzymes that exclude bulk solvent water. If so, an
effective enzymatic “strategy” would be to convert a weak
His hydrogen bond in the Michaelis complex to a strong hy-
Asp 57
102 drogen bond in the transition state, thereby facilitating pro-
CH2 ton transfer while applying the difference in the free en-
O
H2C Ser ergy between the normal and low-barrier hydrogen bonds
C – 195
H N to preferentially binding the transition state. In fact, as
O Perry Frey has shown, the NMR spectrum of the proton
N CH2
linking His 57 to Asp 102 in chymotrypsin (which exhibits
H O
a particularly large downfield chemical shift indicative of
deshielding) is consistent with the formation of an LBHB
His in the transition state (see Fig. 15-25b; the pK’s of proto-
Asp 57 nated His 57 and Asp 102 are nearly equal in the anhy-
102
O CH2 drous environment of the active site complex). This pre-
H2C Ser sumably promotes proton transfer from Ser 195 to His 57
C 195 as in the charge relay mechanism. Moreover, an ultrahigh
N
O H (0.78 Å) resolution X-ray structure of Bacillus lentus
N CH2
–
subtilisin by Richard Bott reveals that the hydrogen bond
H O between His 64 and Asp 32 of its catalytic triad has an un-
"Charge relay system" usually short N p O distance of 2.62  0.01 Å and that its
H atom is nearly centered between the N and O atoms
In the process, the anionic charge of Asp 102 was thought (note that this highly accurate protein X-ray structure is
to be transferred, via a tautomeric shift of His 57, to Ser one of the very few in which H atoms are observed and in
195. The catalytic triad was therefore originally named the which short D p A distances are confidently measured).
charge relay system. It is now realized, however, that such Although several studies, such as the foregoing, have
a mechanism is implausible because an alkoxide ion revealed the existence of unusually short hydrogen bonds
(pK  15) has far greater proton affinity than does His 57 in enzyme active sites, it is far more difficult to demon-
(pK L 7, as measured by NMR techniques). How, then, strate experimentally that they are unusually strong, as
can Asp 102 nucleophilically activate Ser 195? LBHBs are predicted to be. In fact, several studies of the
A possible solution to this conundrum has been pointed strengths of unusually short hydrogen bonds in organic
out by W.W. Cleland and Maurice Kreevoy and, indepen- model compounds in nonaqueous solutions suggest that
dently, by John Gerlt and Paul Gassman. Proton transfers these hydrogen bonds are not unusually strong. Con-
between hydrogen bonded groups (D¬ H p A) only oc- sequently, a lively debate has ensued as to the catalytic sig-
cur at physiologically reasonable rates when the pK of the nificance of LBHBs. Yet if enzymes do not form LBHBs,
proton donor is no more than 2 or 3 pH units greater than it remains to be explained how, in numerous widely ac-
that of the protonated form of the proton acceptor (the cepted enzymatic mechanisms that we shall encounter, the
height of the kinetic barrier,
G ‡, for the protonation of conjugate base of an acidic group can abstract a proton
an acceptor by a more basic donor increases with the dif- from a far more basic group.
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Section 15–3. Serine Proteases 527

e. Much of a Serine Protease’s Catalytic Activity Arises in the duodenal mucosa, specifically hydrolyzes trypsino-
from Preferential Transition State Binding gen’s Lys 15 ¬ Ile 16 peptide bond, thereby excising its N-
Despite the foregoing, blocking the action of the cat- terminal hexapeptide (Fig. 15-27). This yields the active
alytic triad through the specific methylation of His 57 by enzyme, which has Ile 16 at its N-terminus. Since this ac-
treating chymotrypsin with methyl-p-nitrobenzene sulfo- tivating cleavage occurs at a trypsin-sensitive site (recall
nate that trypsin cleaves after Arg and Lys residues), the small
His amount of trypsin produced by enteropeptidase also cat-
57 alyzes activation, generating more trypsin, etc.; that is,
CH2 trypsinogen activation is autocatalytic.
O Chymotrypsin. Chymotrypsinogen is activated by the spe-
H N1 cific tryptic cleavage of its Arg 15 ¬ Ile 16 peptide bond
+ O2N S O CH3
3 to form
-chymotrypsin (Fig. 15-28). -Chymotrypsin
N
O subsequently undergoes autolysis (self-digestion) to specif-
Methyl-p-nitrobenzene ically excise two dipeptides, Ser 14–Arg 15 and Thr 147–
sulfonate Asn 148, thereby yielding the equally active enzyme
-chy-
motrypsin (heretofore and hereafter referred to as
His chymotrypsin). The biochemical significance of this latter
57 process, if any, is unknown.
CH2 Elastase. Proelastase, the zymogen of elastase, is activated
similarly to trypsinogen by a single tryptic cleavage that
H N1 excises a short N-terminal polypeptide.
+ O2N SO3–
3
N+
b. Biochemical “Strategies” That Prevent Premature
CH3 Zymogen Activation
yields an enzyme that is a reasonably good catalyst: It en- Trypsin activates pancreatic procarboxypeptidases A
hances the rate of proteolysis by as much as a factor of and B and prophospholipase A2 (the action of phospho-
2  106 over the uncatalyzed reaction, whereas the native lipase A2 is outlined in Section 25-1) as well as the
enzyme has a rate enhancement factor of
1010. Similarly, pancreatic serine proteases. Premature trypsin activation
the mutation of Ser 195, His 57, or even all three residues can consequently trigger a series of events that lead to
of the catalytic triad yields enzymes that enhance proteo- pancreatic self-digestion. Nature has therefore evolved an
lysis rates by
5  104-fold over that of the uncatalyzed re- elaborate defense against such inappropriate trypsin acti-
action. Evidently, the catalytic triad provides a nucleophile vation. We have already seen (Section 15-3D) that pan-
and is an alternate source and sink of protons (general acid– creatic trypsin inhibitor binds essentially irreversibly to any
base catalysis). However, a large portion of chymotrypsin’s trypsin formed in the pancreas so as to inactivate it.
rate enhancement must be attributed to its preferential bind- Furthermore, the trypsin-catalyzed activation of trypsino-
ing of the catalyzed reaction’s transition state. gen (Fig. 15-27) occurs quite slowly, presumably because
the unusually large negative charge of its highly evolu-
tionarily conserved N-terminal hexapeptide repels the Asp
E. Zymogens at the back of trypsin’s specificity pocket. Finally, pancre-
Most proteolytic enzymes are biosynthesized as somewhat atic zymogens are stored in intracellular vesicles called
larger inactive precursors known as zymogens (enzyme
precursors, in general, are known as proenzymes). In the
case of digestive enzymes, the reason for this is clear: If
+ 10 15 16
these enzymes were synthesized in their active forms, they H3N Val (Asp)4 Lys Ile Val ...
would digest the tissues that synthesized them. Indeed,
acute pancreatitis, a painful and sometimes fatal condition Trypsinogen
that can be precipitated by pancreatic trauma, is charac-
enteropeptidase or
terized by the premature activation of the digestive en- trypsin
zymes synthesized by this gland.
+ ...
a. Serine Proteases Are Autocatalytically Activated H3N Val (Asp)4 Lys + Ile Val
Trypsin, chymotrypsin, and elastase are activated ac- Trypsin
cording to the following pathways:
FIGURE 15-27 Activation of trypsinogen to form trypsin.
Trypsin. The activation of trypsinogen, the zymogen of Proteolytic excision of the N-terminal hexapeptide is catalyzed
trypsin, occurs as a two-stage process when trypsinogen en- by either enteropeptidase or trypsin. The chymotrypsinogen
ters the duodenum from the pancreas. Enteropeptidase, a residue numbering is used here; that is, Val 10 is actually
single-pass transmembrane serine protease that is located trypsinogen’s N-terminus and Ile 16 is trypsin’s N-terminus.
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528 Chapter 15. Enzymatic Catalysis

FIGURE 15-28 Activation of

chymotrypsinogen by proteolytic cleavage. Chymotrypsinogen 136 201 245
1 122
Both - and -chymotrypsin are (inactive)
enzymatically active. See Kinemage S S Cys
Cys Cys
S S Cys
Exercise 10-4
trypsin

Arg Ile
π-Chymotrypsin 1
136 201 245
122
(active) 15 16
S S
S S
chymotrypsin
Ser Arg Thr Asn

14 15 + 147 148

Tyr Ala
Leu Ile
α-Chymotrypsin 1
136 146 149
201 245
13 122
(active) 16
S S
S S

zymogen granules whose membranous walls are thought or greatly relieved numerous human ailments. Such med-
to be resistant to enzymatic degradation. ications include antibiotics (which have enormously re-
duced the impact of infectious diseases), anti-inflammatory
c. Zymogens Have Distorted Active Sites agents (which reduce the effects of inflammatory diseases
Since the zymogens of trypsin, chymotrypsin, and elas- such as arthritis), analgesics and anesthetics (which make
tase have all their catalytic residues, why aren’t they en- modern surgical techniques possible), agents that reduce
zymatically active? Comparisons of the X-ray structures of the incidence and severity of cardiovascular disease and
trypsinogen with that of trypsin and of chymotrypsinogen stroke, antidepressants, antipsychotics, agents that inhibit
with that of chymotrypsin show that on activation, the stomach acid secretion (which prevent stomach ulcers and
newly liberated N-terminal Ile 16 residue moves from the heartburn), agents to combat allergies and asthma, im-
surface of the protein to an internal position, where its free munosuppressants (which make organ transplants possi-
cationic amino group forms an ion pair with the invariant ble), agents used for cancer chemotherapy, and a great
anionic Asp 194 (Fig. 15-21). Aside from this change, how- variety of other substances.
ever, the structures of these zymogens closely resemble Early human cultures almost certainly recognized both
those of their corresponding active enzymes. Surprisingly, the beneficial and toxic effects of indigenous plant and an-
this resemblance includes their catalytic triads, an obser- imal products and used many of them as “medications.”
vation which led to the discovery that these zymogens are Unfortunately, most of these substances were useless or
actually enzymatically active, albeit at a very low level. even harmful. Although there were sporadic attempts over
Careful comparisons of the corresponding enzyme and zy- the 2500 years preceding the modern era to formulate ra-
mogen structures, however, revealed the reason for this tional systems of drug discovery, they had little success be-
low activity: The zymogens’ specificity pockets and oxy- cause they were based mainly on unfounded theories and
anion holes are improperly formed such that, for example, superstition (e.g., the doctrine of signatures stated that if
the amide NH of chymotrypsin’s Gly 193 points in the wrong a plant resembles a particular body part, it must be de-
direction to form a hydrogen bond with the tetrahedral in- signed by nature to influence that body part) rather than
termediate (see Fig. 15-25). Hence, the zymogens’ very low observation and experiment. Consequently, at the begin-
enzymatic activity arises from their reduced ability to bind ning of the 20th century, only three known drugs, apart
substrate productively and to stabilize the tetrahedral in- from folk medicines, were effective in treating specific dis-
termediate. These observations provide further structural eases: (1) Digitalis, a heart stimulant extracted from the
evidence favoring the role of preferred transition state foxglove plant (Section 20-3A), was used to treat various
binding in the catalytic mechanism of serine proteases. heart conditions; (2) quinine (Section 26-4A), obtained
from the bark and roots of the Cinchona tree, was used to
treat malaria; and (3) mercury was used to treat syphilis
(a cure that was often worse than the disease). It was not
4
DRUG DESIGN until several decades later that the rise of the scientific
The improvements in medical care over the past several method coupled to the rapidly increasing knowledge of
decades are, in large measure, attributable to the devel- physiology, biochemistry, and chemistry led to effective
opment of a huge variety of drugs, which have eliminated methods of drug discovery. In fact, the vast majority of
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Section 15–4. Drug Design 529

drugs in use today were discovered and developed in the required to produce a particular toxic effect in animals;
past three decades. and the LD50, the mean lethal dose required to kill 50%
In this section we discuss the elements of drug discov- of a test sample.
ery and pharmacology (the science of drugs, including their For an inhibitor of an enzyme that follows Michaelis-
composition, uses, and effects). The section ends with a Menten kinetics, the IC50 is determined by measuring the
consideration of one of the major successes of modern drug ratio I /o for several values of [I] at constant [S], where
discovery methods, HIV protease inhibitors. I is the initial velocity of the enzyme when the in-
hibitor concentration is [I]. By dividing equation
[14.24] by equation [14.38] with  defined according
A. Techniques of Drug Discovery to equation [14.37], we see that
KM  3 S4 KM  3 S4
Most drugs act by modifying the function of a particular
vI
 
KM  3S4 3I4
receptor in the body or in an invading pathogen. In most [15.13]
KM a1  b  3S4
cases, the receptor is a protein to which the drug specifi- v0
cally binds. It may be an enzyme, a transmembrane chan- K1
nel that transports a specific substance into or out of a cell When I /o  0.5 (50% inhibition),
(Chapter 20), and/or a protein that participates in an inter-
3S 4
3I4  3 IC50 4  KI a1  b
or intracellular signaling pathway (Chapter 19). In all of
[15.14]
these cases, a substance that in binding to a receptor mod- KM
ulates its function is known as an agonist, whereas a sub-
stance that binds to a receptor without affecting its func- Consequently, if the measurements of I /o are made with
tion but blocks the binding of agonists is called an [S]

KM, then [IC50]  KI.
antagonist. The biochemical and physiological effects of a The ratio TD50
ED50 is defined as a drug’s therapeutic
drug and its mechanism of action are referred to as its index, the ratio of the dose of the drug that produces tox-
pharmacodynamics. icity to that which produces the desired effect. It is, of
course, preferable that a drug have a high therapeutic in-
a. Drug Discovery Is a Complex Procedure dex, but this is not always possible.
How are new drugs discovered? Nearly all drugs that
have been in use for over a decade were discovered by b. Cathepsin K Is a Drug Target for Osteoporosis
screening large numbers of synthetic compounds and nat- The development of genomic sequencing techniques
ural products for the desired effect. Drug candidates that (Section 7-2B) and hence the characterization of tens of
are natural products are usually discovered by the frac- thousands of previously unknown genes is providing an
tionation of the organisms in which they occur, which are enormous number of potential drug targets. For example,
often plants used in folk remedies of the conditions of in- osteoporosis, a condition that afflicts postmenopausal
terest. Humans having the condition whose treatment is women and elderly men, is characterized by the progres-
being sought cannot be used as “guinea pigs” in this ini- sive loss of bone mass leading to a greatly increased fre-
tial screening process, and even guinea pigs or other lab- quency of bone fracture, particularly of the hip, spine, and
oratory animals such as mice or dogs (if they can be made wrist. Bones consist of a protein matrix that is 90% type
to be suitable models of the condition under considera- I collagen (Section 8-2B), in which spindle- or plate-shaped
tion) are too expensive to use on the many thousands of crystals of hydroxyapatite, Ca5(PO4)3OH, are embedded.
compounds that are usually tested. Thus, in vitro screens Bones are by no means static structures. They undergo
are initially used, such as the degree of binding of a drug continuous remodeling through the countervailing action
candidate to an enzyme that is implicated in a disease of of two types of bone cells: osteoblasts, which synthesize
interest, toxicity toward the target bacteria in the search bone’s organic matrix in which its mineral component is
for a new antibiotic, or effects on a line of cultured mam- laid down; and osteoclasts, which solubilize mineralized
malian cells. However, as the number of drug candidates bone matrix through the secretion of proteolytic enzymes
is winnowed down, more sensitive screens such as testing into an extracellular bone resorption pit, which is main-
in animals are employed. tained at pH 4.5. The acidic solution dissolves the bone’s
A drug candidate that exhibits a desired effect is called mineral component, thereby exposing its protein matrix to
a lead compound (or, colloquially, a lead). A good lead proteolytic degradation. Osteoporosis arises when bone re-
compound binds to its target receptor with a dissociation sorption outstrips bone formation.
constant, KD
1 M. Such a high affinity is necessary to In the search for a drug target for osteoporosis, a cDNA
minimize a drug’s less specific binding to other macromol- library (Sections 5-5E and 5-5F) was prepared from an
ecules in the body and to ensure that only low doses of the osteoclastoma (a cancer derived from osteoclasts; normally
drug need be taken. For enzyme inhibitors, the dissocia- osteoclasts are very rare cells). Around 4% of these cDNAs
tion constant is the inhibitor’s KI or K¿I (Section 14-3). encode a heretofore unknown protease, which was named
Other common measures of the effect of a drug are the cathepsin K (cathepsins are proteases that occur in the lyso-
IC50, the inhibitor concentration at which an enzyme ex- some). Further studies, both at the cDNA and protein lev-
hibits 50% of its maximal activity; the ED50, the effective els, indicated that cathepsin K is only expressed at high lev-
dose of a drug required to produce a therapeutic effect in els in osteoclasts. Microscopic examination of osteoclasts
50% of a test sample; the TD50, the mean toxic dose that had been stained with antibodies directed against
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530 Chapter 15. Enzymatic Catalysis

cathepsin K revealed that this enzyme is localized at the of a lead compound: For those compounds that had
contact site between osteoclasts and the bone resorption improved efficacy, derivatives were made and tested; etc.
pit. Subsequently, it was shown that mutations in the gene This process has been systematized through the use of
encoding cathepsin K are the cause of pycnodysostosis, a structure–activity relationships (SARs): the determina-
rare hereditary disease which is characterized by hard- tion, via synthesis and screening, of which groups on a lead
ened and fragile bones, short stature, skull deformities, compound are important for its drug function and which
and osteoclasts that demineralize bone normally but are not. For example, if a phenyl group on a lead com-
do not degrade its protein matrix. Evidently, cathepsin pound interacts hydrophobically with a flat region of its
K functions to degrade the protein matrix of bone and receptor, then hydrogenating the phenyl ring to form a
hence is an attractive drug target for the treatment of nonplanar cyclohexane ring will yield a compound with re-
osteoporosis. duced affinity for the receptor.
A logical extension of the SAR concept is to quantify
c. SARs and QSARs Are Useful Tools for it, that is, to determine a quantitative structure–activity re-
Drug Discovery lationship (QSAR). This idea is based on the premise that
A lead compound is used as a point of departure to de- there is a relatively simple mathematical relationship be-
sign more efficacious compounds. Experience has shown tween the biological activity of a drug and its physico-
that even minor modifications to a drug candidate can re- chemical properties. For instance, if the hydrophobicity of
sult in major changes in its pharmacological properties. a drug is important for its biological activity, then changing
Thus, one might place methyl, chloro, hydroxyl, or benzyl the substituents on the drug so as to alter its hydropho-
groups at various places on a lead compound in an effort bicity will affect its activity. A measure of the substance’s
to improve its pharmacodynamics. For most drugs in use hydrophobicity is its partition coefficient, P, between the
today, 5 to 10 thousand related compounds were typically two immiscible solvents, octanol and water, at equilibrium:
synthesized in generating the medicinally useful drug.
These were not random procedures but were guided by ex- concentration of drug in octanol
P [15.15]
perience as medicinal chemists tested various derivatives concentration of drug in water
Biological activity may be expressed as 1
C, where C is the
(a) drug concentration required to achieve a specified level of
biological function (e.g., IC50). Then a plot of log 1
C ver-
sus log P (the use of logarithms keeps the plot on a man-
ageable scale) for a series of derivatives of the lead com-
pound having a relatively small range of log P values often
indicates a linear relationship (Fig. 15-29a), which can
1 therefore be expressed:
log __
C

log a b  k1 log P  k2
1
[15.16]
C
Here k1 and k2 are constants, whose optimum values in this
QSAR can be determined by computerized curve-fitting
methods. For compounds with a larger range of log P val-
log P ues, it is likely that a plot of log 1
C versus log P will have
a maximum value (Fig. 15-29b) and hence be better de-
(b) scribed by a quadratic equation:

log a b  k1 1log P2 2  k2 log P  k3

1
[15.17]
C
Of course, the biological activities of few substances
1
depend only on their hydrophobicities. A QSAR can
log __ therefore simultaneously take into account several physico-
C
chemical properties of substituents such as their pK val-
ues, van der Waals radii, hydrogen bonding energy, and
conformation. The values of the constants for each of the
terms in a QSAR is indicative of the contribution of that
term to the drug’s activity. The use of QSARs to optimize
the biological activity of a lead compound has proven to
log P be a valuable tool in drug discovery.
FIGURE 15-29 Hypothetical QSAR plots of log(1/C) versus
log P for a series of related compounds. (a) A plot that is best d. Structure-Based Drug Design
described by a linear equation. (b) A plot that is best described Since the mid 1980s, dramatic advances in the speed and
by a quadratic equation. precision with which a macromolecular structure can be
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Section 15–4. Drug Design 531

determined by X-ray crystallography and NMR (Section would far more effectively map out the potential range of
8-3A) have enabled structure-based drug design, a process the substituent and possibly identify an unexpectedly ac-
that greatly reduces the number of compounds that need tive analog. Interestingly, QSAR and computational tech-
be synthesized in a drug discovery program. As its name niques have been combined in the development of “virtual
implies, structure-based drug design (also called rational combinatorial chemistry,” a procedure in which libraries of
drug design) uses the structure of a receptor in complex compounds are computationally “synthesized” and “ana-
with a drug candidate to guide the development of more lyzed” to predict their efficacy, thereby again reducing the
efficacious compounds. Such a structure will reveal, for ex- number of compounds that must actually be synthesized
ample, the positions of the hydrogen bonding donors and in order to generate an effective drug.
acceptors in a receptor binding site as well as cavities in
the binding site into which substituents might be placed on
a drug candidate to increase its binding affinity for the re-
B. Introduction to Pharmacology
ceptor. These direct visualization techniques are usually The in vitro development of an effective drug candidate is
supplemented with molecular modeling tools such as the only the first step in the drug development process. Besides
computation of the minimum energy conformation of a causing the desired response in its isolated target receptor,
proposed derivative, quantum mechanical calculations that a useful drug must be delivered in sufficiently high concen-
determine its charge distribution and hence how it would tration to this receptor where it resides in the human body
interact electrostatically with the receptor, and docking without causing unacceptable side effects.
simulations in which an inhibitor candidate is computa-
tionally modeled into the binding site on the receptor to a. Pharmacokinetics Is a Multifaceted Phenomenon
assess potential interactions. Structure-based drug design The most convenient form of drug administration is
is an iterative process: The structure of the receptor in com- orally (by mouth). In order to reach its target receptor, a
plex with a compound with improved properties is deter- drug administered in this way must surmount a series of
mined in an effort to further improve its properties. formidable barriers: (1) It must be chemically stable in the
highly acidic (pH 1) environment of the stomach and must
e. Combinatorial Chemistry and High-Throughput not be degraded by the digestive enzymes in the gastroin-
Screening testinal tract; (2) it must be absorbed from the gastroin-
As structure-based methods were developed, it ap- testinal tract into the bloodstream, that is, it must pass
peared that they would become the dominant mode of drug through several cell membranes; (3) it must not bind too
discovery. However, the recent advent of combinatorial tightly to other substances in the body (e.g., lipophilic sub-
chemistry techniques to rapidly and inexpensively synthe- stances tend to be absorbed by certain plasma proteins and
size large numbers of related compounds combined with by fat tissue; anions may be bound by plasma proteins,
the development of robotic high-throughput screening mainly albumin; and cations may be bound by nucleic
techniques has caused the drug discovery “pendulum” to acids); (4) it must survive derivatization by the battery of
again swing toward the “make-many-compounds-and-see- enzymes, mainly in the liver, that function to detoxify
what-they-do” approach. A familiar example of combina- xenobiotics (foreign compounds), as discussed below (note
torial chemistry is the parallel synthesis of the large that the intestinal blood flow drains directly into the liver
number of different oligonucleotides on a DNA chip via the portal vein, so that the liver processes all orally in-
(Section 7-6B). Similarly, if a lead compound can be syn- gested substances before they reach the rest of the body);
thesized in a stepwise manner from several smaller mod- (5) it must avoid rapid excretion by the kidneys; (6) it must
ules, then the substituents on each of these modules can pass from the capillaries to its target tissue; (7) if it is tar-
be varied in parallel to produce a library of related geted to the brain, it must cross the blood–brain barrier,
compounds (e.g., Fig. 15-30). which blocks the passage of most polar substances; and
A variety of synthetic techniques have been developed (8) if it is targeted to an intracellular receptor, it must pass
that permit the combinatorial synthesis of thousands of re- through the plasma membrane and, possibly, other intra-
lated compounds in a single procedure. Thus, whereas in- cellular membranes. The ways in which a drug interacts
vestigations into the importance of a hydrophobic group with these various barriers is known as its pharmacoki-
at a particular position in a lead compound might previ- netics. Thus, the bioavailability of a drug (the extent to
ously have prompted the individual syntheses of only the which it reaches its site of action, which is usually taken to
ethyl, propyl, and benzyl derivatives of the compound, the be the systemic circulation) depends on both the dose given
use of combinatorial synthesis would permit the genera- and its pharmacokinetics. Of course, barriers (1) and (2)
tion of perhaps 100 different groups at that position. This can be circumvented by injecting the drug [e.g., some forms

R2 R2
O H
R1 N R2CHO R1 N R3NH2 FIGURE 15-30 The combinatorial synthesis of
N
R1 N R3 arylidene diamides. If ten different variants of
O O H each R group are used in the synthesis, then 1000
O
O O different derivatives will be synthesized.
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532 Chapter 15. Enzymatic Catalysis

of penicillin (Fig. 11-25) must be injected because their Phase II. This phase mainly tests the efficacy of the
functionally essential
-lactam rings are highly susceptible drug against the target disease in 100 to 500 volunteer pa-
to acid hydrolysis], but this mode of drug delivery is tients but also refines the dosage range and checks for side
undesirable for long-term use. effects. The effects of the drug candidate are usually as-
Since the pharmacokinetics of a drug candidate is as sessed via single blind tests, procedures in which the pa-
important to its efficacy as is its pharmacodynamics, both tient is unaware of whether he/she has received the drug
must be optimized in producing a medicinally useful drug. or a control substance. Usually the control substance is a
The following empirically based rules, formulated by placebo (an inert substance with the same physical ap-
Christopher Lipinski and known as Lipinski’s “rule of pearance, taste, etc., as the drug being tested) but, in the
five,” state that a compound is likely to exhibit poor case of a life-threatening disease, it is an ethical necessity
absorption or permeation if: that the control substance be the best available treatment
against the disease.
1. Its molecular mass is greater than 500 D.
Phase III. This phase monitors adverse reactions from
2. It has more than 5 hydrogen bond donors (expressed
long-term use as well as confirming efficacy in 1000 to 5000
as the sum of its OH and NH groups).
patients. It pits the drug candidate against control sub-
3. It has more than 10 hydrogen bond acceptors (ex- stances through the statistical analysis of carefully designed
pressed as the sum of its N and O atoms). double blind tests, procedures in which neither the patients
4. Its value of log P is greater than 5. nor the clinical investigators evaluating the patients’ re-
sponses to the drug know whether a given patient has
Drug candidates that disobey Rule 1 are likely to have low received the drug or a control substance. This is done to
solubilities and to only pass through cell membranes with minimize bias in the subjective judgments the investigators
difficulty; those that disobey Rules 2 and/or 3 are likely to must make.
be too polar to pass through cell membranes; and those
that disobey Rule 4 are likely to be poorly soluble in aque- Currently, only about 5 drug candidates in 5000 that en-
ous solution and hence unable to gain access to membrane ter preclinical trials reach clinical trials. Of these, only one,
surfaces. Thus, the most effective drugs are usually a com- on average, is ultimately approved for clinical use, with
promise; they are neither too lipophilic nor too hydrophilic.
40% of drug candidates passing Phase I trials and
50%
In addition, their pK values are usually in the range 6 to 8 of those passing Phase II trials (most drug candidates that
so that they can readily assume both their ionized and un- enter Phase III trials are successful). In recent years, the
ionized forms at physiological pH’s. This permits them to preclinical portion of a drug discovery process has aver-
cross cell membranes in their unionized form and to bind aged
3 years to complete, whereas successful clinical tri-
to their receptor in their ionized form. However, since the als have usually required an additional 7 to 10 years. These
concentration of a drug at its receptor depends, as we saw, successive stages of the drug discovery process are in-
on many different factors, the pharmacokinetics of a drug creasingly expensive, so that to successfully bring a drug
candidate may be greatly affected by even small chemical to market costs, on average, around $300 million.
changes. QSARs and other computational tools have been The most time-consuming and expensive aspect of a
developed to predict these effects but they are, as yet, drug development program is identifying a drug candi-
rather crude. date’s rare adverse reactions. Nevertheless, it is not an un-
common experience for a drug to be brought to market
only to be withdrawn some months or years later when it
b. Toxicity and Adverse Reactions Eliminate Most
is found to have caused unanticipated life-threatening side
Drug Candidates
effects in as few as 1 in 10,000 individuals (the search for
The final criteria that a drug candidate must meet are
new applications of an approved drug and its postmarket-
that its use be safe and efficacious in humans. Tests for
ing surveillance are known as its Phase IV clinical trials).
these properties are initially carried out in animals, but
For example, in 1997, the FDA withdrew its approval of
since humans and animals often react quite differently to
the drug fenfluramine (fen),
a particular drug, the drug must ultimately be tested in hu-
mans through clinical trials. In the United States, clinical
trials are monitored by the Food and Drug Administration CH3
(FDA) and have three increasingly detailed (and expen- CH2 CH NH CH2 CH3
sive) phases: CH3

Phase I. This phase is primarily designed to test the CH2 C NH2

safety of a drug candidate but is also used to determine its
CF3 CH3
dosage range and the optimal dosage method (e.g., orally
vs injection) and frequency. It is usually carried out on a Fenfluramine Phentermine
small number (20–100) of normal, healthy volunteers, but
in the case of a drug candidate known to be highly toxic which it had approved in 1973 for use as an appetite sup-
(e.g., a cancer chemotherapeutic agent), it is carried out pressant in short-term (a few weeks) weight loss programs.
on volunteer patients with the target disease. Fenfluramine had become widely prescribed, often for ex-
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Section 15–4. Drug Design 533

tended periods, together with another appetite suppres-

sant, phentermine (phen; approved in 1959), a combination
known as fen-phen (although the FDA had not approved
of the use of the two drugs in combination, once it ap-
proves a drug for any purpose, a physician may prescribe
it for any other purpose). The withdrawal of fenfluramine
was prompted by over 100 reports of heart valve damage
in individuals (mostly woman) who had taken fen-phen for
an average of 12 months (phentermine was not withdrawn
because the evidence indicated that fenfluramine was the
responsible agent). This rare side effect had not been ob-
served in the clinical trials of fenfluramine, in part because,
being an extremely unusual type of drug reaction, it had
not been screened for.

c. The Cytochromes P450 Metabolize Most Drugs

Why is it that a drug that is well tolerated by the ma-
jority of patients can pose such a danger to others? FIGURE 15-31 X-Ray structure of cytochrome P450CAM from
Differences in reactions to drugs arise from genetic differ- Pseudomonas putida showing its active site region. The heme
ences among individuals as well as differences in their group, the Cys side chain that axially ligands its Fe atom, and
disease states, other drugs they are taking, age, sex, and en- the enzyme’s lipophilic substrate thiocamphor are shown in
vironmental factors. The cytochromes P450, which func- ball-and-stick form with N blue, O red, S yellow, Fe orange, and
the C atoms of the heme, its liganding Cys side chain, and the
tion in large part to detoxify xenobiotics and participate in
thiocamphor green, cyan, and pale blue-green, respectively. The
the metabolic clearance of the majority of drugs in use, bonds liganding the Fe are gray. [Based on an X-ray structure by
provide instructive examples of these phenomena. Thomas Poulos, University of California at Irvine. PDBid 8CPP.]
The cytochromes P450 constitute a superfamily of
heme-containing enzymes that occur in nearly all living or-
ganisms, from bacteria to mammals [their name arises from gated (covalently linked) to polar substances such as glu-
the characteristic 450-nm peak in their absorption spectra curonic acid (Section 11-1C), glycine, sulfate, and acetate,
when reacted in their Fe(II) state with CO]. Humans ex- which further enhances aqueous solubility. The many types
press 100 isozymes (catalytically and structurally similar of cytochromes P450 in animals, which have different sub-
but genetically distinct enzymes from the same organism; strate specificities (although these specificities tend to be
also called isoforms) of cytochromes P450, mainly in the broad and hence often overlap), are thought to have arisen
liver but also in other tissues (its various isozymes are in response to the numerous toxins which plants produce,
named by the letters “CYP” followed by a number desig- presumably to discourage animals from eating them.
nating its family, an uppercase letter designating its sub- Drug–drug interactions are often mediated by cyto-
family, and often another number; e.g., CYP2D6). These chromes P450. For example, if drug A is metabolized by
monooxygenases (Fig. 15-31), which in animals are em- or otherwise inhibits a cytochrome P450 isozyme that me-
bedded in the endoplasmic reticulum membrane, catalyze tabolizes drug B, then coadministering drugs A and B will
reactions of the sort cause the bioavailability of drug B to increase above the
value it would have had if it alone had been administered.
RH  O2  2H   2e ∆ ROH  H 2O This phenomenon is of particular concern if drug B has a
low therapeutic index. Conversely, if, as is often the case,
The electrons (e) are supplied by NADPH, which passes drug A induces the increased expression of the cytochrome
them to cytochrome P450’s heme prosthetic group via the P450 isozyme that metabolizes it and drug B, then co-
intermediacy of the enzyme cytochrome P450 reductase. administering drugs A and B will reduce drug B’s bioavail-
Here RH represents a wide variety of usually lipophilic ability, a phenomenon that was first noted when certain
compounds for which the different cytochromes P450 are antibiotics caused oral contraceptives to lose their efficacy.
specific. They include polycyclic aromatic hydrocarbons Moreover, if drug B is metabolized to a toxic product, its
[PAHs, frequently carcinogenic (cancer-causing) com- increased rate of reaction may result in an adverse reac-
pounds that are present in tobacco smoke, broiled meats, tion. Environmental pollutants such as PAHs or PCBs are
and other pyrolysis products], polycyclic biphenyls (PCBs, also known to induce the expression of specific cytochrome
which were widely used in electrical insulators and as plas- P450 isozymes and thereby alter the rates at which certain
ticizers and are also carcinogenic), steroids (in whose syn- drugs are metabolized. Finally, some of these same effects
theses cytochromes P450 participate; Sections 25-6A and may occur in patients with liver disease, as well as arising
25-6C), and many different types of drugs. The xenobiotics from age-based, gender-based, and individual differences
are thereby converted to a more water-soluble form, which in liver physiology.
aids in their excretion by the kidneys. Moreover, the newly Although the cytochromes P450 presumably evolved to
generated hydroxyl groups are often enzymatically conju- detoxify and/or help eliminate harmful substances, in sev-
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534 Chapter 15. Enzymatic Catalysis

O O O FIGURE 15-32 The metabolic

reactions of acetaminophen that
H C HO C C convert it to its conjugate with
N CH3 N CH3 N CH3
glutathione.

cytochrome P450 spontaneous

O2 H2O H2O
OH OH O
Acetaminophen Acetimidoquinone
(N-acetyl-p-aminophenol)

SH

COO– O CH2 O
+
H3N CH CH2 CH2 C NH CH C NH CH2 COO–
Glutathione
(-L-Glutamyl-L-cysteinyl-glycine)

H C
N CH3

S
OH
COO– O CH2 O
+
H3N CH CH2 CH2 C NH CH C NH CH2 COO–
Acetaminophen–glutathione conjugate

eral cases they have been shown to participate in convert- rates of drug metabolism have been characterized for many
ing relatively innocuous compounds to toxic agents. For of the cytochromes P450. The distributions of these vari-
example, acetaminophen (Fig. 15-32), a widely used anal- ous alleles differ markedly among ethnic groups and hence
gesic and antipyretic (fever reducer), is quite safe when probably arose to permit each group to cope with the tox-
taken in therapeutic doses (1.2 g/day for an adult) but in ins in its particular diet.
large doses (10 g) is highly toxic. This is because, in ther- Polymorphism in a given cytochrome P450 results in
apeutic amounts, 95% of the acetaminophen present is en- differences between individuals in the rates at which they
zymatically glucuronidated or sulfated at its ¬ OH group metabolize certain drugs. For instance, in cases that a cy-
to the corresponding conjugates, which are readily ex- tochrome P450 variant has absent or diminished activity,
creted. The remaining 5% is converted, through the action otherwise standard doses of a drug that the enzyme nor-
of a cytochrome P450 (CYP2E1), to acetimidoquinone mally metabolizes may cause the bioavailability of the drug
(Fig. 15-32), which is then conjugated with glutathione, a to reach toxic levels. Conversely, if a particular P450
tripeptide with an unusual -amide bond that participates enzyme has enhanced activity (usually because the gene
in a wide variety of metabolic processes (Section 26-4C). encoding it has been duplicated one or more times), higher
However, when acetaminophen is taken in large amounts, than normal doses of a drug that the enzyme metabolizes
the glucuronidation and sulfation pathways become satu- would have to be administered to obtain the required ther-
rated and hence the cytochrome P450-mediated pathway apeutic effect. However, if the drug is metabolized to a
becomes increasingly important. If hepatic (liver) gluta- toxic product, this may result in an adverse reaction.
thione is depleted faster than it can be replaced, acetimi- Several known P450 variants have altered substrate speci-
doquinone, a reactive compound, instead conjugates with ficities and hence produce unusual metabolites, which also
the sulfhydryl groups of cellular proteins, resulting in of- may cause harmful side effects.
ten fatal hepatotoxicity. Experience has amply demonstrated that there is no
Many of the cytochromes P450 in humans are unusu- such thing as a drug that is entirely free of adverse reactions.
ally polymorphic, that is, there are several common alleles However, as the enzymes and their variants that partici-
(variants) of the genes encoding these enzymes. Alleles pate in drug metabolism are characterized and rapid and
that cause diminished, enhanced, and qualitatively altered inexpensive genotyping methods are developed, it may be-
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Section 15–4. Drug Design 535

come possible to tailor drug treatment to an individual’s infection (during most of which the host exhibits no
genetic makeup rather than to the population as a whole. obvious symptoms), the host’s immune system is steadily
depleted until it has deteriorated to the point that the host
regularly falls victim to and is eventually killed by oppor-
C. HIV Protease and Its Inhibitors
tunistic pathogens that individuals with normally func-
Acquired immunodeficiency syndrome (AIDS), the only tioning immune systems can readily withstand. It is this
major epidemic attributable to a previously unknown latter stage of an HIV infection that is called AIDS. In the
pathogen to appear in the 20th century (it was first de- absence of effective therapy, AIDS is almost invariably fa-
scribed in 1981), is caused by human immunodeficiency tal. Through the year 2002, an estimated 30 million people
virus type 1 (HIV-1; the closely related HIV-2, which we had died of AIDS and an estimated 42 million others,
shall not explicitly discuss here, also causes AIDS and has largely in sub-Saharan Africa, were HIV-positive, numbers
a similar response to drugs). HIV-1, which was discovered that are increasing at the rate of
5 million per year. As a
in 1983, is a retrovirus, a family of viruses that were inde- consequence of this global catastrophe, HIV has been char-
pendently characterized in 1970 by David Baltimore and acterized and effective countermeasures against it have
Howard Temin. The retroviral genome is a single-stranded been devised faster than for any other pathogen in history.
RNA that reproduces inside its host cell by transcribing
the RNA to double-stranded DNA in a process mediated a. Reverse Transcriptase Inhibitors Are Only
by the virally encoded enzyme reverse transcriptase Partially Effective
(Section 30-4C). The DNA is then inserted into the host The first drug to be approved by the FDA (in 1987)
cell’s chromosomal DNA by a viral enzyme named to fight AIDS was 3-azido-3-deoxythymidine (AZT;
integrase and is passively replicated along with the cell’s zidovudine),
DNA. However, under activating conditions (which for
HIV-1 often is an infection by another pathogen), the HOCH2 T
O
retroviral DNA is transcribed, the proteins it encodes are
expressed and inserted in or anchored to the host cell H H
H H
plasma membrane, and new virions (virus particles) are _
 +
produced by the budding out of a viral protein-laden seg- N N N H
ment of plasma membrane so as to enclose viral RNA (Fig. 3-Azido-3-deoxythymidine
15-33). (AZT; zidovudine)
HIV-1 is targeted to and specifically replicates within
helper T cells, essential components of the immune system which had first been synthesized in 1964 as a possible anti-
(Section 35-2A). Unlike most types of retroviruses, HIV-1 cancer agent (it was ineffective). AZT is a nucleoside ana-
eventually kills the cells producing it. Although the helper log that, on enzymatic conversion to its triphosphate in the
T cells within which HIV-1 are actively replicating are of- cell (the plasma membrane is impermeable to nucleoside
ten destroyed by the immune system, those within which triphosphates), inhibits HIV-1 reverse transcriptase, as do
the HIV-1 is latent (its DNA is not being transcribed) are the several other drugs (Section 30-4C) that the FDA had
not detected by the immune system and hence provide a approved to treat AIDS prior to 1996. Unfortunately, these
reservoir of HIV-1 (other types of cells also harbor HIV-1). agents only slow the progression of an HIV infection but
Consequently, over a several year period after the initial do not stop it. This is in part because they are toxic, mainly

FIGURE 15-33 The assembly, budding,

and maturation of HIV-1. SU is the
surface glycoprotein gp120 and TM is
the transmembrane protein gp41.
[After Turner, B.G. and Summers, M.F.,
J. Mol. Biol. 285, 4 (1999).]
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536 Chapter 15. Enzymatic Catalysis

(a)
(a)
I II III IV

MA CA p1 NC p6

gag

I II III V VI VII VIII

MA CA p1 TF PR RT RN IN

gag–pol

(b)
Cleavage Sequence
site
... Ser -Gln-Asn- Tyr — Pro - Ile - Val - Gln ...
I
FIGURE 15-34 HIV-1 polyproteins. (a) The organization of ... Ala -Arg- Val -Leu — Ala -Glu- Ala -Met ...
II
the HIV-1 gag and gag–pol polyproteins. The symbols used are ... Ala - Thr - Ile -Met — Met-Gln-Arg- Gly ...
III
MA, matrix protein; CA, capsid protein; NC, nucleocapsid ... Pro - Gly -Asn- Phe — Leu-Gln- Ser -Arg ...
IV
protein; TF, transmembrane protein; PR, protease; RT, reverse ... Ser -Phe-Asn- Phe — Pro -Gln- Ile - Thr ...
V
transcriptase; RN, ribonuclease; and IN, integrase. (b) The ... Thr -Leu-Asn- Phe — Pro - Ile - Ser - Pro ...
VI
sequences flanking the HIV-1 protease cleavage sites (red ... Ala -Glu- Thr - Phe — Tyr - Val -Asp- Gly ...
VII
bonds) indicated in Part a. ... Arg- Lys - Ile -Leu — Phe -Leu-Asp- Gly ...
VIII

to the bone marrow cells that are blood cell precursors, these enzymes all contain catalytically essential Asp
and hence cannot be taken in large doses. More important, residues that occur in the signature sequence Asp–
however, is that reverse transcriptase, unlike most other Thr/Ser–Gly. Humans have several known aspartic pro-
DNA polymerases (Section 30-2A), cannot correct its mis- teases including pepsin, a digestive enzyme secreted by the
takes and hence frequently generates mutations (about one stomach (its specificity is indicated in Table 7-2) that func-
per 104 bp and, since the viral genome consists of
104 bp, tions at pH 1 and which was the first enzyme to be recog-
each viral genome bears, on average, one new mutation). nized (named in 1825 by T. Schwann); chymosin (formerly
Consequently, under the selective pressure of an anti-HIV rennin), a stomach enzyme, occurring mainly in infants,
drug such as AZT, the drug’s target receptor rapidly evolves that specifically cleaves a Phe–Met peptide bond in the
to a drug-resistant form. milk protein -casein, thereby causing milk to curdle, mak-
ing it easier to digest (calf stomach chymosin has been used
for millennia to make cheese); cathepsins D and E, lyso-
b. HIV-1 Polyproteins Are Cleaved by HIV-1 Protease
somal proteases that function to degrade cellular proteins;
HIV-1, as do other retroviruses, synthesizes its proteins
renin, which participates in the regulation of blood pres-
in the form of polyproteins, which each consist of several
sure and electrolyte balance (Fig. 15-35); and -secretase
tandemly linked proteins (Fig. 15-34). HIV-1 encodes two
(also known as memapsin 2), a transmembrane protein
polyproteins, gag (55 kD) and gag–pol (160 kD), which are
common in brain that participates in cleaving A precur-
both anchored to the plasma membrane via N-terminal
sor protein to yield amyloid- protein (A), which is im-
myristoylation (Section 12-3B). These polyproteins are
plicated in Alzheimer’s disease (Section 9-5B). In addition,
then cleaved to their component proteins through the ac-
many fungi secrete aspartic proteases, presumably to aid
tion of HIV-1 protease, but only after this enzyme has ex-
them in invading the tissues they colonize.
cised itself from gag–pol. This process occurs only after the
Eukaryotic aspartic proteases are
330-residue mono-
virion has budded off from the host cell and results in a
meric proteins. The X-ray structure of pepsin (Fig. 15-36a),
large structural reorganization of the virion (Fig. 15-33).
which closely resembles those of other eukaryotic aspartic
The virion is thereby converted from its noninfectious im-
proteases, reveals that this croissant-shaped protein con-
mature form to its pathogenic mature form. If HIV-1 pro-
sists of two homologous domains that are related by
tease is inactivated, either mutagenically or by an inhibitor,
approximate 2-fold symmetry (although only about 25
the virion remains noninfectious. Hence HIV-1 protease is
residues in the core  sheets of each domain are closely
an opportune drug target.
related by this symmetry). Each domain contains a cat-
alytically essential Asp in an analogous position. The X-
c. Aspartic Proteases and Their Catalytic Mechanism ray structures of enzyme–inhibitor complexes of various
HIV-1 protease is a member of the aspartic protease aspartate proteases indicate that substrates bind in a
family (also known as acid proteases), so called because prominent cleft between the two domains that could ac-
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Section 15–4. Drug Design 537

1
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His13 What is the catalytic mechanism of eukaryotic aspartic
Angiotensinogen proteases? Proteolytic enzymes, in general, have three es-
sential catalytic components:
H2O renin
1. A nucleophile to attack the carbonyl C atom of the
scissile peptide to form a tetrahedral intermediate (Ser 195
1
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu10 + Val-Ile-His
serves this function in trypsin; Fig. 15-23).
Angiotensin I
2. An electrophile to stabilize the negative charge that
H2O angiotensin converting enzyme (ACE) develops on the carbonyl O atom of the tetrahedral inter-
mediate (the H-bonding donors lining the oxyanion hole,
1
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe8 + His-Leu Gly 193 and Ser 195, do so in trypsin; Fig. 15-25).
Angiotensin II 3. A proton donor so as to make the amide N atom of
the scissile peptide a good leaving group (the imidazolium
FIGURE 15-35 Renin participation in blood pressure group of His 57 in trypsin; Fig. 15-23).
regulation. Renin proteolytically cleaves the 13-residue
polypeptide angiotensinogen to the 10-residue polypeptide Pepsin’s pH rate profile (Section 14-4) suggests that it
angiotensin I. This latter peptide is then cleaved by angiotensin has two ionizable essential residues, one with pK L 1.1
converting enzyme (ACE) to the 8-residue polypeptide and the other with pK L 4.7, which are almost certainly the
angiotensin II, which, on binding to its receptor, induces carboxyl groups of its essential Asp residues. At the pH of
vasoconstriction and retention of Na
and water by the
the stomach, the Asp residue with pK 4.7 is protonated
kidneys, resulting in increased blood pressure. Consequently
there have been considerable efforts to develop both renin and
and that with pK 1.1 is partially ionized. This suggests that
ACE inhibitors for the control of hypertension (high blood the ionized carboxyl group acts as a nucleophile to form
pressure), although as yet, only ACE inhibitors have been the putative tetrahedral intermediate. However, no cova-
approved as drugs. lent intermediate between an aspartic protease and its sub-
strate has ever been detected.
The two active site Asp residues in eukaryotic aspartic
proteases are in close proximity and both appear to form
hydrogen bonds to a bridging water molecule that is pres-
commodate an
8-residue polypeptide segment in an ex- ent in several X-ray structures of eukaryotic aspartic pro-
tended  sheetlike conformation. The active site Asp teases (Fig. 15-36b). This, together with a variety of enzy-
residues are located at the base of this cleft (Fig. 15-36a). mological and kinetic data, led Thomas Meeks to propose

(a) (b)
FIGURE 15-36 X-Ray structure of pepsin. (a) Ribbon diagram viewer. (b) Enlarged view of the active site Asp residues and
in which the N-terminal domain (residues 1–172) is gold, the their bound water molecule indicating the lengths (in Å) of
C-terminal domain (residues 173–326) is cyan, the side chains possible hydrogen bonds (thin gray bonds). The X-ray structures
of the active site Asp residues are shown in ball-and-stick form of other aspartic proteases exhibit similar interatomic distances.
with C green and O red, and the water molecule that is bound [Based on an X-ray structure by Anita Sielecki and Michael
by these Asp side chains is represented by a large red sphere. James, University of Alberta, Edmonton, Canada. PDBid
The protein is viewed with the pseudo-2-fold axis relating core 4PEP.]
portions of the two domains tipped from vertical toward the
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538 Chapter 15. Enzymatic Catalysis

the following catalytic mechanism for aspartic proteases d. HIV-1 Protease Inhibitors Are Effective
(Fig. 15-37): Anti-AIDS Agents
HIV-1 protease differs from eukaryotic aspartic pro-
1. An active site Asp carboxylate group, acting as a
teases in that it is a homodimer of 99-residue subunits.
general base, activates the bound water molecule, the so-
Nevertheless, its X-ray structure (Fig. 15-38a), determined
called lytic water, to nucleophilically attack the scissile pep-
independently in 1989 by Alexander Wlodawer, by Manual
tide’s carbonyl C as an OH ion. Proton donation (general
Navia and Paula Fitzgerald, and by Tom Blundell, closely
acid catalysis) by the second, previously uncharged active
resembles those of eukaryotic aspartic proteases. Thus,
site Asp stabilizes the oxyanion that would otherwise form
HIV-1 protease has the enzymatically unusual property
in the resulting tetrahedral intermediate.
that its single active site is formed by two identical sym-
2. The N atom of the scissile peptide is protonated by
the first Asp (general acid catalysis) resulting, through
charge rearrangement and proton transfer to the second
Asp (general base catalysis), in amide bond scission.
Aspartic proteases are inhibited by compounds with tetra-
hedral carbon atoms at a position mimicking a scissile pep-
tide bond (see below). This strongly suggests that these
enzymes preferentially bind their transition states (transi-
tion state stabilization), thereby enhancing catalysis.

H
H
R N
C R R N
C R
O O (a)
1
H H H H
O O O –O O O

C H H
– C C C
O O O O

Asp Asp Asp Asp

Michaelis complex Tetrahedral intermediate

R H R
C O + N
O H H (b)
H
FIGURE 15-38 X-Ray structure of HIV-1 protease.
O O
(a) Uncomplexed and (b) in complex with its inhibitor
C C saquinavir (structural formula in Fig. 15-41). In each structure,
O –O the homodimeric protein is viewed with its 2-fold axis of
symmetry vertical and is shown as a ribbon diagram with one
Asp Asp subunit gold and the other cyan. The side chains of the active
site Asp residues, Asp 25 and Asp 25, as well as the saquinavir
Products
in Part b, are shown in ball-and-stick form with C green, N
FIGURE 15-37 Catalytic mechanism of aspartic proteases. blue, and O red. Note how the  hairpin “flaps” at the top of
(1) The nucleophilic attack of the enzyme-activated water the uncomplexed enzyme have folded down over the inhibitor
molecule (red) on the carbonyl carbon atom of the scissile in the saquinavir complex. Compare these structures with that
peptide bond (green) to form the tetrahedral intermediate. This of the similarly viewed pepsin in Fig. 15-36a. [Part a based on
reaction step is promoted by general base catalysis by the Asp an X-ray structure by Tom Blundell, Birkbeck College, London,
on the right and general acid catalysis by the Asp on the left U.K., and Part b based on an X-ray structure by Robert
(blue). (2) The decomposition of the tetrahedral intermediate Crowther, Hoffmann-LaRoche Ltd., Nutley, New Jersey.
to form products via general acid catalysis by the Asp on the PDBids (a) 3PHV and (b) 1HXB.]
right and general base catalysis by the Asp on the left. See the Interactive Exercises
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Section 15–4. Drug Design 539

metrically arranged subunits. Quite possibly HIV-1 pro- 2-fold symmetric enzyme in a two-fold pseudosymmetric
tease resembles the putative primordial aspartic protease extended conformation such that the inhibitor interacts
that, through gene duplication, evolved to form the eu- with the enzyme much like a strand in a  sheet (Fig.
karyotic enzymes (although HIV-1 protease is well suited 15-39). On the “floor” of the binding cleft, each signature
to the limited amount of genetic information that a virus sequence (Asp 25–Thr 26–Gly 27) is located in a loop that
can carry). is stabilized by a network of hydrogen bonds similar to that
Once the structure of HIV-1 protease became available, observed in eukaryotic aspartic proteases. The inhibitor in-
intensive efforts were mounted in numerous laboratories teracts with the enzyme via a hydrogen bond to the active
to find therapeutically effective inhibitors of this enzyme. site residue Asp 25. However, contrary to the case for eu-
In this process,
200 X-ray structures and several NMR karyotic aspartic proteases (Fig. 15-36b), no X-ray struc-
structures have been reported of HIV-1 protease, its mu- ture of an HIV-1 protease contains a water molecule within
tants, and the proteases of other retroviruses, both alone hydrogen bonding distance of Asp 25 or Asp 25. On the
and in their complexes with a great variety of inhibitors. flap side of the binding cleft, the inhibitor interacts with
Hence, HIV-1 protease is perhaps the most exhaustively Gly 48 and Gly 48 and with a water molecule that is not
structurally studied protein. the attacking nucleophile but which mediates the contacts
Comparison of the X-ray structure of HIV-1 protease between the flaps and the inhibitor backbone.
alone (Fig. 15-38a) with that of its complexes with polypep- Although HIV-1 protease specifically cleaves the gag
tidelike inhibitors (e.g., Fig. 15-38b) reveals that, on bind- and gag–pol polyproteins at a total of 8 sites (Fig. 15-34b),
ing an inhibitor, the  hairpin “flaps” covering the “top” these sites appear to have little in common except that their
of the substrate-binding cleft move down by as much as immediately flanking residues are nonpolar and mostly
7 Å to enclose the inhibitor. Such an inhibitor binds to the bulky. Indeed, binding studies indicate that HIV-1 pro-

Flap side
S2
S4 S1 S3

P2
P4 P1 P3

Ile 50 Ile 50

N N
Gly 48 H H Gly 48 Gly 48
N C C N
H H
O
O H H
O

O O O O
H H H H
C N C N C N C N
C N C N C N C N
H H H H
O O O O
O O
H O HO H
O N C O OC N O
Asp 29
O Asp 29 Asp 29 Gly 27 Gly 27 O
Asp 25 Asp 25

P3 P1 P2 P4

S3 S1 S2 S4
FIGURE 15-39 Arrangement of hydrogen bonds between HIV-1 designated P1, P2, P3, … , counting toward the C-terminus;
protease and a modeled substrate. In the nomenclature used here, and the symbols S1, S2, S3, … , and S1, S2, S3, … , designate the
polypeptide residues in one subunit are assigned primed numbers enzyme’s corresponding residue-binding subsites. The scissile
to differentiate them from the residues of the other subunit; peptide bond is marked by arrows. [After Wlodawer, A. and
substrate residues on the N-terminal side of the scissile peptide Vondrasek, J., Annu. Rev. Biophys. Biomol. Struct. 27, 257
bond are designated P1, P2, P3, … , counting toward the (1998).]
N-terminus; substrate residues on its C-terminal side are
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540 Chapter 15. Enzymatic Catalysis

O R
Ph H OH
N OH
H
N C CH N N
CH N C N •H2SO4
H O
O N O
Peptide Bond H
Indinavir (CrixivanTM)
R

H
N CH2 CH
CH N C
H
R O S O NHtBu
O
Reduced Amide
HO •CH3SO3H
N N
OH R
H
H H OH
N CH CH H
CH CH2 C

R O Nelfinavir (ViraceptTM)
Hydroxyethylene Ph
N H OH H CH3
OH R
O
H O N N N
N CH CH S N
CH CH C O O N
H
Ph
R OH O S
Ritonavir (NorvirTM)
Dihydroxyethylene

OH O
H H
N CH N C H O NHtBu
CH CH2 CH O
N
R R
N N N •CH3SO3H
H
Hydroxyethylamine O OH
NH2 O H
FIGURE 15-40 Comparison of a normal peptide bond (top) to H
a selection of groups (red) that are isosteres (stereochemical
analogs) of the tetrahedral intermediate in reactions catalyzed Saquinavir (InviraseTM)
by aspartic proteases.

H OH NH2

tease’s specificity arises from the cumulative effects of the O N N

S
interactions between the enzyme and the amino acids in O O
O
positions P4 through P¿4 . However, three of the peptides O
Ph
cleaved by HIV-1 have either the sequence Phe-Pro or Tyr- Amprenavir (AgeneraseTM)
Pro, which are sequences that human aspartic proteases do
not cleave. Hence, HIV-1 protease inhibitors containing FIGURE 15-41 Some HIV-1 protease inhibitors that are in
clinical use. Note that in addition to its generic (chemical)
groups that resemble either of these dipeptides would be
name, each drug has a proprietary trade name, here in
unlikely to inhibit essential human aspartic proteases. parentheses, under which it is marketed.
An effective HIV-1 protease inhibitor should resemble
a substrate with its scissile peptide replaced by a group that
the enzyme cannot cleave. Such a group should, prefer-
ably, enhance the enzyme’s affinity for the inhibitor.
Mimics of the tetrahedral intermediate (Fig. 15-37), that proteases) and pharmacokinetics (they do not readily pass
is, transition state analogs, are likely to do so. Conse- through cell membranes.). Consequently, therapeutically
quently, a variety of such groups (Fig. 15-40) have been in- effective HIV-1 protease inhibitors must be peptido-
vestigated in efforts to synthesize therapeutically effective mimetics (peptide mimics), substances that sterically and
inhibitors of HIV-1 protease. perhaps physically, but not chemically, resemble polypep-
Although HIV-1 protease has high in vitro affinity for tides. The use of peptidomimetics also permits conforma-
its polypeptide-based inhibitors, these substances have tional constraints to be imposed on a drug candidate that
poor oral bioavailability (they are degraded by digestive would not be present in the corresponding polypeptide.
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Chapter Summary 541

As of early 2003, the FDA had approved six HIV-1 pro- tease inhibitor to improve the latter’s pharmacokinetics.
tease inhibitors (Fig. 15-41), the first of which, saquinavir, The plasma virus levels in many patients who were
was sanctioned in late 1995. These peptidomimetics have placed on combination therapy rapidly became unde-
IC50s against HIV in culture ranging from 2 to 60 nM but tectable and have remained so for several years. This, how-
have little or no activity against human aspartic proteases ever, does not constitute a cure: If drug therapy is inter-
(KI’s 10 M). They are the first drugs to clearly prolong rupted, the virus will reappear in the plasma because
the lives of AIDS victims. Their development, in each case, certain tissues in the body harbor latent viruses that are
was a complex iterative process that required the design, unaffected by and/or inaccessible to drug therapy. Thus,
synthesis, and evaluation of numerous related compounds. the presently available anti-HIV medications must be
In several cases, these investigations capitalized on the taken for a lifetime.
wealth of experience gained in developing peptidomimetic Current anti-HIV therapies are by no means ideal. To
inhibitors of the aspartic protease renin and in the result- maximize their oral bioavailability, some of the different
ing stockpiles of these compounds. drugs must be taken well before or after a meal but oth-
All the FDA-approved HIV-1 protease inhibitors ini- ers must be taken with a meal. To minimize the probabil-
tially cause a rapid and profound decline in a patient’s ity of resistant forms of HIV arising, the bioavailability of
plasma HIV load, which is often paralleled by immune each drug must be maintained at a certain minimum level
system recovery. However, as we saw with reverse tran- and hence each drug must be taken on a rigid schedule.
scriptase inhibitors, mutant forms of the protease that are Moreover, these drugs have significant side effects, mainly
resistant to the inhibitor being used arise, usually within fatigue, nausea, diarrhea, tingling and numbness with ri-
4 to 12 weeks. Moreover, such a mutant protease is likely tonavir, and kidney stones with indinavir. Consequently,
to be resistant to other HIV-1 protease inhibitors, because numerous AIDS patients fail to take their medications
all of the HIV-1 protease inhibitors are targeted to the properly, which greatly increases the likelihood that they
same binding site. This has led to the use of combination will develop resistance to these drugs and infect others with
therapies in which an HIV-1 protease inhibitor is adminis- drug-resistant viruses. Finally, HIV-1 protease inhibitors,
tered together with one, or more often, two reverse tran- being complex molecules, are difficult to synthesize and
scriptase inhibitors. This is because any virus that gains re- therefore are relatively expensive, so that in the develop-
sistance to one drug in a regimen will be suppressed by the ing countries in which AIDS is most prevalent, govern-
other drug(s) in that regimen. In addition, the HIV-1 pro- ments and most individuals cannot afford to purchase these
tease inhibitor ritonavir has been shown to be a potent in- drugs, even if they were to be supplied at cost. It is
hibitor of the cytochrome P450 isoforms (CYP3A4,5,7) therefore important that anti-HIV therapies be developed
that metabolize other protease inhibitors and hence is usu- that are easy for patients to comply with, are inexpensive,
ally prescribed in low dosage as an adjunct to another pro- and ideally, will totally eliminate an HIV infection.

CHAPTER SUMMARY
1
Catalytic Mechanisms Most enzymatic mechanisms substrates in a bimolecular reaction arrests their relative mo-
of catalysis have ample precedent in organic catalytic reac- tions resulting in a rate enhancement. The preferential enzy-
tions. Acid- and base-catalyzed reactions occur, respectively, matic binding of the transition state of a catalyzed reaction
through the donation or abstraction of a proton to or from a over the substrate is an important rate enhancement mecha-
reactant so as to stabilize the reaction’s transition state com- nism. Transition state analogs are potent competitive in-
plex. Enzymes often employ ionizable amino acid side chains hibitors because they bind to the enzyme more tightly than
as general acid–base catalysts. Covalent catalysis involves nu- does the corresponding substrate.
cleophilic attack of the catalyst on the substrate to transiently 2
Lysozyme Lysozyme catalyzes the hydrolysis of
form a covalent bond followed by the electrophilic stabiliza- (1S4)-linked poly(NAG–NAM), the bacterial cell wall
tion of a developing negative charge in the reaction’s transi- polysaccharide, as well as that of poly(NAG). According to
tion state. Various protein side chains as well as certain coen- the Phillips mechanism, lysozyme binds a hexasaccharide so
zymes can act as covalent catalysts. Metal ions, which are as to distort its D-ring toward the half-chair conformation of
common enzymatic components, catalyze reactions by stabi- the planar oxonium ion transition state. This is followed by
lizing developing negative charges in a manner resembling cleavage of the C1 ¬ O1 bond between the D- and E-rings as
general acid catalysis. Metal ion–bound water molecules are promoted by proton donation from Glu 35. Finally, the re-
potent sources of OH ions at neutral pH’s. Metal ions also sulting oxonium ion transition state is electrostatically stabi-
facilitate enzymatic reactions through the charge shielding of lized by the nearby carboxyl group of Asp 52 so that the E-ring
bound substrates. The arrangement of charged groups about can be replaced by OH to form the hydrolyzed product. The
an enzymatic active site of low dielectric constant in a man- roles of Glu 35 and Asp 52 in lysozyme catalysis have been
ner that stabilizes the transition state complex results in the verified through mutagenesis studies. Similarly, structural and
electrostatic catalysis of the enzymatic reaction. Enzymes cat- binding studies indicate that strain is of major catalytic im-
alyze reactions by bringing their substrates into close prox- portance in the lysozyme mechanism. However, mass spec-
imity in reactive orientations. The enzymatic binding of the trometry and X-ray studies have demonstrated that the
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542 Chapter 15. Enzymatic Catalysis

lysozyme reaction proceeds via a covalent glycosyl–enzyme tested for drug efficacy in an assay that is a suitable surrogate
intermediate involving Asp52 rather than by the noncova- of the disease/condition under consideration. Lead com-
lently bound oxonium ion intermediate postulated by the pounds are then chemically manipulated in the search for
Phillips mechanism. compounds with improved drug efficacy. Structure–activity
3
Serine Proteases Serine proteases constitute a wide- relationships (SARs) and quantitative structure–activity
spread class of proteolytic enzymes that are characterized by relationships (QSARs) are useful tools in this endeavor.
the possession of a reactive Ser residue. The pancreatically Structure-based drug design uses the X-ray and NMR struc-
synthesized digestive enzymes trypsin, chymotrypsin, and tures of drug candidates in complex with their target proteins,
elastase are sequentially and structurally related but have dif- together with a variety of molecular modeling tools, to guide
ferent side chain specificities for their substrates. All have the the search for improved drug candidates. However, the advent
same catalytic triad, Asp 102, His 57, and Ser 195, at their ac- of combinatorial chemistry and high-throughput screening
tive sites. The differing side chain specificities of trypsin and procedures has extended the “make-many-compounds-and-
chymotrypsin depend in a complex way on the structures of see-what-they-do” approaches to drug discovery. In order to
the loops that connect the walls of the specificity pocket, as reach their target receptors, drugs must have favorable phar-
well as on the charge of the side chain at the base of the speci- macokinetics, that is, they must readily traverse numerous
ficity pocket. Subtilisin, serine carboxypeptidase II, and ClpP physical barriers in the body, avoid chemical transformation
are unrelated serine proteases that have essentially the same by enzymes, and not be excreted too rapidly. Most useful drugs
active site geometry as do the pancreatic enzymes. Catalysis are neither too lipophilic nor too hydrophilic so that they can
in serine proteases is initiated by the nucleophilic attack of both gain access to the necessary membranes and pass through
the active Ser on the carbonyl carbon atom of the scissile pep- them. Drug toxicity, dosage, efficacy, and the nature of rare
tide to form the tetrahedral intermediate, a process that may adverse reactions are determined through extensive and care-
be facilitated by the formation of a low-barrier hydrogen bond fully designed clinical trials. Most drugs are metabolically
between Asp 102 and His 57. The tetrahedral intermediate, cleared through oxidative hydroxylation by one of the
100
which is stabilized by its preferential binding to the enzyme’s cytochrome P450 isozymes. This permits the hydroxylated
active site, then decomposes to the acyl–enzyme intermediate drugs to be enzymatically conjugated to polar groups such as
under the impetus of proton donation from the Asp 102- glucuronic acid and glycine, which increases their rates of ex-
polarized His 57. After the replacement of the leaving group cretion by the kidneys. Drug–drug interactions are frequently
by solvent H2O, the catalytic process is reversed to yield the mediated by cytochromes P450. Polymorphisms among cy-
second product and the regenerated enzyme. The Asp tochromes P450 are often responsible for the variations
102–His 57 couple therefore functions in the reaction as a pro- among individuals in their response to a given drug, includ-
ton shuttle. The active Ser is not unusually reactive but is ide- ing adverse reactions.
ally situated to nucleophilically attack the activated scissile The formulation of HIV-1 protease inhibitors to control
peptide. The X-ray structure of the trypsin–BPTI complex in- HIV infections is one of the major triumphs of modern drug
dicates the existence of the tetrahedral intermediate, whereas discovery methods. HIV are retroviruses that attack specific
X-ray structures of a complex of elastase with the heptapep- immune system cells and thereby degrade the immune system
tide BCM7 have visualized both the acyl–enzyme intermedi- over a period of several years to the point that it is no longer
ate and the tetrahedral intermediate. able defend against opportunistic infections. HIV-1 protease
The pancreatic serine proteases are synthesized as zymo- functions to cleave the polyproteins in immature HIV-1 viri-
gens to prevent pancreatic self-digestion. Trypsinogen is acti- ons that have budded out from a host cell, thus generating the
vated by a single proteolytic cleavage by enteropeptidase. The mature, infectious form. HIV-1 protease is an aspartic pro-
resulting trypsin similarly activates trypsinogen as well as chy- tease that, as eukaryotic aspartic proteases such as pepsin, uses
motrypsinogen, proelastase, and other pancreatic digestive its two active site Asp residues to activate its bound lytic wa-
enzymes. Trypsinogen’s catalytic triad is structurally intact. ter molecule as the nucelophile that attacks and thereby
The zymogen’s low catalytic activity arises from a distortion cleaves specific peptide bonds in the substrate polyprotein.
of its specificity pocket and oxyanion hole, so that it is unable All of the FDA-approved peptidomimetic inhibitors of HIV-
to productively bind substrate or preferentially bind the cat- 1 protease cause a rapid and profound decrease in plasma HIV
alytic reaction’s transition state. levels, although they do not entirely eliminate the virus. They
4
Drug Design Drugs act by binding to and thereby are used in combination with reverse transcriptase inhibitors
modifying the functions of receptors. Many promising drug to minimize the ability of the rapidly mutating HIV to evolve
candidates, which are known as lead compounds, have been drug-resistant forms.
found by methods in which a large number of compounds are

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PROBLEMS
1. Explain why
-pyridone is not nearly as effective a catalyst 2. RNA is rapidly hydrolyzed in alkaline solution to yield a
for glucose mutarotation as is -pyridone. What about -pyri- mixture of nucleotides whose phosphate groups are bonded to ei-
done? ther the 2 or the 3 positions of the ribose residues. DNA, which
7884d_c15.qxd 1/23/03 12:29 PM Page 545 mac18 mac18:df_169:7884D:

Problems 545

lacks RNA’s 2 OH groups, is resistant to alkaline degradation. 4. In the following lactonization reaction,
Explain.
O O
3. Carboxypeptidase A, a Zn2 -containing enzyme, hydro- OH
C
lyzes the C-terminal peptide bonds of polypeptides (Section
7-1A). In the enzyme–substrate complex, the Zn2 ion is coordi- 
CH2
nated to three enzyme side chains, the carbonyl oxygen of the C CH2 COO C
scissile peptide bond, and a water molecule. A plausible model R R R R R R
for the enzyme’s reaction mechanism that is consistent with X-
ray and enzymological data is diagrammed in Fig. 15-42. What the relative reaction rate when R  CH 3 is 3.4  1011 times that
are the roles of the Zn2 ion and Glu 270 in this mechanism? when R  H. Explain.
*5. Derive the analog of Eq. [15.11] for an enzyme that cat-
alyzes the reaction:
A BSP
Assume the enzyme must bind A before it can bind B:
E A B ∆ EA B ∆ EAB S EP
CO2–
6. Explain, in thermodynamic terms, why an “enzyme” that
O CHR stabilizes its Michaelis complex as much as its transition state does
Glu 270 C not catalyze a reaction.
NH R
O– 7. Suggest a transition state analog for proline racemase that
C O differs from those discussed in the text. Justify your suggestion.
H
O 8. Wolfenden has stated that it is meaningless to distinguish
2+
between the “binding sites” and the “catalytic sites” of enzymes.
H Zn
Explain.
9. Explain why oxalate (OOCCOO) is an inhibitor of ox-
Michaelis complex
aloacetate decarboxylase.
attack of
water
10. In light of the information given in this chapter, why are
enzymes such large molecules? Why are active sites almost al-
ways located in clefts or depressions in enzymes rather than on
protrusions?
CO2–
O 11. Predict the effects on lysozyme catalysis of changing Phe
CHR 34, Ser 36, and Trp 108 to Arg, assuming that this change does
Glu 270 C
NH R not significantly alter the structure of the protein.
O–
C *12. The incubation of (NAG)4 with lysozyme results in the
H
slow formation of (NAG)6 and (NAG)2. Propose a mechanism
O O– for this reaction. What aspect of the Phillips mechanism is estab-
H Zn2+ lished by this reaction?
13. How would the lysozyme binding affinity of the following
(1S4)-linked tetrasaccharide
Tetrahedral intermediate
scissle bond CH2OH
scission H O
H
NAG NAM NAG O H
CO2– H

CHR
H NHCOCH3
N
O compare with that of NAG–NAM–NAG–NAM? Explain.
H H
Glu 270 C
+ 14. A major difficulty in investigating the properties of the
O– pancreatic serine proteases is that these enzymes, being proteins
R
themselves, are self-digesting. This problem is less severe, how-
C O
ever, for solutions of chymotrypsin than it is for solutions of
O trypsin or elastase. Explain.
H Zn2+ 15. The comparison of the active site geometries of chy-
motrypsin and subtilisin under the assumption that their similar-
ities have catalytic significance has led to greater mechanistic
Enzyme-product complex
understanding of both these enzymes. Discuss the validity of this
FIGURE 15-42 Mechanism of carboxypeptidase A. strategy.
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546 Chapter 15. Enzymatic Catalysis

16. Benzamidine (KI  1.8  105M) and leupeptin (KI  is an inhibitor of subtilisin and chymotrypsin. Indicate the struc-
1.8  107M) ture of these enzyme–inhibitor complexes.
18. Tofu (bean curd), a high-protein soybean product that is
O O widely consumed in China and Japan, is prepared in such a way
CH3C Leu Leu NH CH CH as to remove the trypsin inhibitor present in soybeans. Explain
the reason(s) for this treatment.
(CH2)3
19. Explain why mutating all three residues of trypsin’s
NH catalytic triad has essentially no greater effect on the enzyme’s
catalytic rate enhancement than mutating only Ser 195.
C C
H2N NH2+ H2N NH2+ 20. Explain why chymotrypsin is not self-activating as is
Benzamidine Leupeptin trypsin.
21. Does Lipinski’s “rule of five” predict that a hexapeptide
are both specific competitive inhibitors of trypsin. Explain their would be a therapeutically effective drug? Explain.
mechanisms of inhibition. Design leupeptin analogs that inhibit
22. The preferred antidote for acetaminophen overdose is
chymotrypsin and elastase.
N-acetylcysteine. Explain why the administration of this sub-
17. Trigonal boronic acid derivatives have a high tendency to stance, which must occur within 8 to 16 hours of the overdose, is
form tetrahedral adducts. 2-Phenylethyl boronic acid an effective treatment.
OH 23. Why would the activation of HIV-1 protease before the
virus buds from its host cell be disadvantageous to the virus?
CH2 CH2 B Explain.
OH
2–Phenylethyl boronic acid