Voet D., Voet Ju.G. Biochemistry

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serine proteases. The primordial trypsin gene evidently

arose before the divergence of prokaryotes and eukary-

otes.

There are three known serine proteases whose primary

and tertiary structures bear no discernible relationship to

each other or to chymotrypsin but which, nevertheless,

contain catalytic triads at their active sites whose structures

closely resemble that of chymotrypsin:

1. Subtilisin, an endopeptidase that was originally iso-

lated from Bacillus subtilis.

2. Wheat germ serine carboxypeptidase II, an exopepti-

dase whose structure is surprisingly similar to that of car-

boxypeptidase A (Fig. 8-19a) even though the latter pro-

tease has an entirely different catalytic mechanism from

that of the serine proteases (see Problem 3).

3. E. coli ClpP, which functions in the degradation of

cellular proteins (Section 32-6B).

Since the orders of the corresponding active site residues

in the amino acid sequences of the four types of serine

proteases are quite different (Fig. 15-22), it seems highly

improbable that they could have evolved from a common

ancestor serine protease. These proteins apparently consti-

tute a remarkable example of convergent evolution: Nature

seems to have independently discovered the same catalytic

mechanism at least four times. (In addition, human cy-

tomegalovirus protease, an essential protein for virus

replication that bears no resemblance to the above

proteases, has active site Ser and His residues whose rela-

tive positions are similar to those in other serine proteases

but lacks an active site Asp residue; it appears to have a

catalytic dyad.)

C. Catalytic Mechanism

See Guided Exploration 12: The catalytic mechanism of serine

proteases

The extensive active site homologies among the

various serine proteases indicate that they all have the

same catalytic mechanism. On the basis of considerable

chemical and structural data gathered in many laborato-

ries, the following catalytic mechanism has been formu-

lated for the serine proteases, here given in terms of chy-

motrypsin (Fig. 15-23):

1. After chymotrypsin has bound substrate to form

the Michaelis complex, Ser 195, in the reaction’s rate-deter-

mining step, nucleophilically attacks the scissile peptide’s

carbonyl group to form a complex known as the tetrahe-

dral intermediate (covalent catalysis). X-ray studies indi-

cate that Ser 195 is ideally positioned to carry out this nu-

cleophilic attack (proximity and orientation effects). The

imidazole ring of His 57 takes up the liberated proton,

thereby forming an imidazolium ion (general base cataly-

sis). This process is aided by the polarizing effect of the

unsolvated carboxylate ion of Asp 102, which is hydrogen

bonded to His 57 (electrostatic catalysis; see Section 15-

3Dd). Indeed, the mutagenic replacement of trypsin’s Asp

Section 15-3. Serine Proteases 531

Figure 15-22 Relative positions of the active site residues in

subtilisin, chymotrypsin, serine carboxypeptidase II, and ClpP

protease. The peptide backbones of Ser 214,Trp 215, and Gly 216

in chymotrypsin, and their counterparts in subtilisin, participate

Ser 125

Leu 126

Gly 127

Ser 221

Subtilisin

His 64

Asp 32

COO

–

Ser 214

Trp 215

Gly 216

Chymotrypsin

Ser 195

His 57

COO

–

Asp

102

Ser

146

Serine

carboxypeptidase II

COO

–

His

397

Asp

338

Ser

Asp

171

His

122

ClpP

protease

COO

–

in substrate-binding interactions. [After Robertus, J.D.,Alden,

R.A., Birktoft, J.J., Kraut, J., Powers, J.C., and Wilcox, P.E.,

Biochemistry 11, 2449 (1972).]

See Kinemage Exercise 10-2

JWCL281_c15_506-556.qxd 2/19/10 9:28 PM Page 531

102 by Asn leaves the enzyme’s K

substantially un-

changed at neutral pH but reduces its k

cat

to ⬃0.05% of

its wild-type value. Neutron diffraction studies have

demonstrated that Asp 102 remains a carboxylate ion rather

than abstracting a proton from the imidazolium ion to form

an uncharged carboxylic acid group. The tetrahedral

532 Chapter 15. Enzymatic Catalysis

R⬘

Enzyme–substrate

complex

Nucleophilic

attack

Asp

102

....

His

Ser

195

–

Tetrahedral intermediate

–

R⬘

Asp

102

....

His

Ser

195

–

Acyl–enzyme intermediate

New N-terminus of

cleaved polypeptide

chain

Substrate

polypeptide

R⬘NH

N CH

Asp

102

....

His

–

Ser

195

R⬘

Asp

102

....

His

Ser

195

–

New C-terminus

of cleaved polypeptide

chain

Active enzymeTetrahedral intermediate

Asp

102

....

His

Ser

195

–

Asp

102

....

His

–

Ser

195

Figure 15-23 Catalytic mechanism of the serine proteases. The

reaction involves (1) the nucleophilic attack of the active site Ser

on the carbonyl carbon atom of the scissile peptide bond to form

the tetrahedral intermediate; (2) the decomposition of the

tetrahedral intermediate to the acyl–enzyme intermediate

through general acid catalysis by the active site Asp-polarized

His, followed by loss of the amine product and its replacement by

a water molecule; (3) the reversal of Step 2 to form a second

tetrahedral intermediate; and (4) the reversal of Step 1 to yield

the reaction’s carboxyl product and the active enzyme.

JWCL281_c15_506-556.qxd 2/19/10 9:28 PM Page 532

intermediate has a well-defined, although transient, exis-

tence. We shall see that much of chymotrypsin’s catalytic

power derives from its preferential binding of the transi-

tion state leading to this intermediate (transition state bind-

ing catalysis).

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 (R¿NH

, 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

serine proteases has prompted numerous investigations of

its validity. In this section we discuss several of the most re-

vealing of these studies.

a. The Tetrahedral Intermediate Is Mimicked in a

Complex of Trypsin with Trypsin Inhibitor

Convincing structural evidence for the existence of the

tetrahedral intermediate was provided by Robert Huber in

an X-ray study of the complex between the 58-residue pro-

tein bovine pancreatic trypsin inhibitor (BPTI) and

trypsin. BPTI binds to and inactivates trypsin, thereby pre-

venting any trypsin that is prematurely activated in the

pancreas from digesting that organ (Section 15-3E). BPTI

binds to the active site region of trypsin across a tightly

packed interface that is cross-linked by a complex network

of hydrogen bonds. This complex’s 10

1

association

constant, among the largest of any known protein–protein

interaction, emphasizes BPTI’s physiological importance.

The portion of BPTI in contact with the trypsin active

site resembles bound substrate. The side chain of BPTI Lys

15I (here “I” differentiates BPTI residues from trypsin

residues) occupies the trypsin specificity pocket (Fig.15-24a)

and the peptide bond between Lys 15I and Ala 16I is posi-

tioned as if it were the scissile peptide bond (Fig. 15-24b).

What is most remarkable about this structure is that its ac-

tive site complex assumes a conformation well along the re-

action coordinate toward the tetrahedral intermediate: The

side chain oxygen of trypsin Ser 195, the active Ser, is in

closer-than-van der Waals contact (2.6 Å) with the pyrami-

dally distorted carbonyl carbon of BPTI’s “scissile” peptide.

Despite this close contact, the proteolytic reaction cannot

proceed past this point along the reaction coordinate be-

cause of the rigidity of the active site complex and because

it is so tightly sealed that the leaving group cannot leave

and water cannot enter the reaction site.

Protease inhibitors are common in nature, where they

have protective and regulatory functions. For example, cer-

tain plants release protease inhibitors in response to insect

bites, thereby causing the offending insect to starve by in-

activating its digestive enzymes. Protease inhibitors consti-

tute ⬃10% of the nearly 200 proteins of blood serum. For

instance, ␣

-proteinase inhibitor, which is secreted by the

liver, inhibits leukocyte elastase (leukocytes are a type of

white blood cell; the action of leukocyte elastase is thought

to be part of the inflammatory process). Pathological vari-

ants of 

-proteinase inhibitor with reduced activity are as-

sociated with pulmonary emphysema, a degenerative dis-

ease of the lungs resulting from the hydrolysis of its elastic

fibers. Smokers also suffer from reduced activity of their



-proteinase inhibitor because of the oxidation of its ac-

tive site Met residue. Full activity of this inhibitor is not re-

gained until several hours after smoking.

Section 15-3. Serine Proteases 533

Figure 15-24 Trypsin–BPTI complex. (a) The X-ray structure

shown as a cutaway surface drawing indicating how trypsin (red)

binds BPTI (green).The green protrusion extending into the red

cavity near the center of the figure represents the Lys 15I side

chain occupying trypsin’s specificity pocket. Note the close

complementary fit of these two proteins. [Courtesy of Michael

Connolly, New York University.] (b) Trypsin Ser 195, the active

Ser, is in closer-than-van der Waals contact with the carbonyl

carbon of BPTI’s scissile peptide, which is pyramidally distorted

toward Ser 195.The normal proteolytic reaction is apparently

arrested somewhere along the reaction coordinate between the

Michaelis complex and the tetrahedral intermediate.

Ser 195

␣

Ala 16I

Lys 15I

(b)

(a)

JWCL281_c15_506-556.qxd 2/19/10 9:28 PM Page 533

b. Serine Proteases Preferentially Bind the

Transition State

Detailed comparisons of the X-ray structures of several

serine protease–inhibitor complexes have revealed a

further structural basis for catalysis in these enzymes

(Fig. 15-25):

1. The conformational distortion that occurs with the

formation of the tetrahedral intermediate causes the car-

bonyl oxygen of the scissile peptide to move deeper into

the active site so as to occupy a previously unoccupied po-

sition, the oxyanion hole.

2. There it forms two hydrogen bonds with the enzyme

that cannot form when the carbonyl group is in its normal

trigonal conformation. These two enzymatic hydrogen

bond donors were first noted by Joseph Kraut to occupy

corresponding positions in chymotrypsin and subtilisin. He

proposed the existence of the oxyanion hole based on the

premise that convergent evolution had made the active

sites of these unrelated enzymes functionally identical.

3. The tetrahedral distortion, moreover, permits the for-

mation of an otherwise unsatisfied hydrogen bond between

the enzyme and the backbone NH group of the residue pre-

ceding the scissile peptide. Consequently, the enzyme binds

the tetrahedral intermediate in preference to either the

Michaelis complex or the acyl–enzyme intermediate.

It is this phenomenon that is responsible for much of the

catalytic efficiency of serine proteases (see below). In fact,

the reason that DIPF is such an effective inhibitor of serine

proteases is because its tetrahedral phosphate group

makes this compound a transition state analog of the

enzyme.

c. The Tetrahedral Intermediate and the Water

Molecule Attacking the Acyl–Enzyme Intermediate

Have Been Directly Observed

Most enzymatic reactions turn over far too rapidly for

their intermediate states to be studied by X-ray or NMR

techniques. Consequently, much of our structural knowl-

edge of these intermediate states derives from the study of

enzyme–inhibitor complexes or complexes of substrates

with inactivated enzymes. Yet the structural relevance of

these complexes is subject to doubt precisely because they

are catalytically unproductive.

In an effort to rectify this situation for serine pro-

teases, Janos Hajdu and Christopher Schofield searched

for peptide–protease complexes that are stable at a pH at

which the protease is inactive but that could be rendered

active by changing the pH. To do so, they screened libraries

of peptides for their ability to bind to porcine pancreatic

elastase at pH 3.5 (at which His 57 is protonated and hence

unable to act as a general base) through the use of ESI-MS

(Section 7-1I). They thereby discovered that YPFVEPI, a

heptapeptide segment of the human milk protein ␤-casein

that is named BCM7, forms a complex with elastase, whose

mass is consistent with the formation of an ester linkage

between BCM7 and the enzyme. In the presence of

at pH 7.5 (where elastase is active), the

O label was incor-

porated into both BCM7 and the elastase–BCM7 complex,

thereby demonstrating that the reaction of BCM7 with

elastase is reversible at this pH. Fragmentation studies by

fast atom bombardment–tandem mass spectrometry

(FAB–MS/MS; Section 7-1I) further revealed that BCM7

that had been incubated with elastase in the presence of

at pH 7.5 incorporated the

O label into only its

C-terminal Ile residue.

534 Chapter 15. Enzymatic Catalysis

Figure 15-25 Transition state stabilization in the serine

proteases. (a) In the Michaelis complex, the trigonal carbonyl

carbon of the scissile peptide is conformationally constrained

from binding in the oxyanion hole (upper left). (b) In the

tetrahedral intermediate, the now charged carbonyl oxygen of

the scissile peptide (the oxyanion) has entered the oxyanion

hole, thereby hydrogen bonding to the backbone NH groups of

NHNH

Oxyanion hole

Ser 195

His 57

Asp

102

Gly 193

R C

CNHN

R′

(a)

Ser 195

His 57

Asp

102

R C

R′

(b)

+NHHN

–

Gly 193

...

Gly 193

.....

...

.. ..

–

Gly 193 and Ser 195.The consequent conformational distortion

permits the NH group of the residue preceding the scissile

peptide bond to form an otherwise unsatisfied hydrogen bond to

Gly 193. Serine proteases therefore preferentially bind the

tetrahedral intermediate. [After Robertus, J.D., Kraut, J., Alden,

R.A., and Birktoft, J.J., Biochemistry 11, 4302 (1972).]

See

Kinemage Exercise 10-3

JWCL281_c15_506-556.qxd 2/19/10 9:28 PM Page 534

The X-ray structure of the BCM7–elastase complex at

pH 5 (Fig. 15-26a) revealed that BCM7’s C-terminal car-

boxyl group, in fact, forms an ester linkage with elastase’s

Ser 195 side chain hydroxyl group to form the expected

acyl–enzyme intermediate. Moreover, this X-ray structure

reveals the presence of a bound water molecule that appears

poised to nucleophilically attack the ester linkage (the

distance from this water molecule to BCM7’s C-terminal

C atom is 3.1 Å and the line between them is nearly per-

pendicular to the plane of the acyl group). His 57, which is

hydrogen bonded to this water molecule, is properly posi-

tioned to abstract one of its protons, thereby activating it

for the nucleophilic attack (general base catalysis).The car-

bonyl O atom of the acyl group occupies the enzyme’s

oxyanion hole such that it is hydrogen bonded to the main

chain N atoms of both Ser 195 and Gly 193. This is in agree-

ment with spectroscopic measurements indicating that the

acyl–enzyme intermediate’s carbonyl group is, in fact,

hydrogen bonded to the oxyanion hole. It was initially

assumed that the oxyanion hole acts only to stabilize the

tetrahedral oxyanion transition state that resides near

the tetrahedral intermediate on the catalytic reaction coor-

dinate. However, it now appears that the oxyanion hole

also functions to polarize the carbonyl group of the

acyl–enzyme intermediate toward an oxyanion (electro-

static catalysis).

The catalytic reaction was initiated in crystals of the

BCM7–elastase complex by transferring them to a buffer at

pH 9.After soaking in this buffer for 1 min,the crystals were

rapidly frozen in liquid N

(196°C), thereby arresting the

enzymatic reaction (recall that the catalytically essential

collective motions of proteins cease at such low tempera-

tures; Section 9-4a). The X-ray structure of such a frozen

crystal (Fig. 15-26b) revealed that the above acyl–enzyme

intermediate had converted to the tetrahedral intermedi-

ate, whose oxyanion, as expected, remained hydrogen

bonded to the N atoms of Ser 195 and Gly 193. Comparison

of this crystal structure with that of the acyl–enzyme inter-

mediate reveals that the enzyme’s active site residues do

not significantly change their positions in the conversion

from the acyl–enzyme intermediate to the tetrahedral inter-

mediate. However, the peptide substrate must do so out of

steric necessity when the trigonal planar acyl group con-

verts to the tetrahedral oxyanion (compare Figs. 15-26a and

15-26b). In response, several enzyme residues that contact

the peptide but which are distant from the active site also

shift their positions (not shown in Fig. 15-26).

d. The Role of the Catalytic Triad: Low-Barrier

Hydrogen Bonds

The earlier literature postulated that the Asp 102-

polarized His 57 side chain directly abstracts a proton

Section 15-3. Serine Proteases 535

Figure 15-26 X-ray structures of porcine pancreatic elastase in

complex with the heptapeptide BCM7 (YPFVEPI). The residues

of elastase are specified by the three-letter code and those of

BCM7 are specified by the one-letter code. (a) The complex at

pH 5.The enzyme’s active site residues and the heptapeptide

(whose N-terminal three residues are disordered) are shown in

ball-and-stick form with elastase C green, BCM7 C cyan, N blue,

O red, S yellow, and the bond between the Ser 195 O atom and

the C-terminal C atom of BCM7 lavender.The enzyme-bound

water molecule, which appears poised to nucleophilically attack

the acyl–enzyme’s carbonyl C atom, is represented by an orange

sphere. The dashed gray lines represent the catalytically

important hydrogen bonds and the dotted gray line indicates the

trajectory that the bound water molecule presumably follows in

nucleophilically attacking the acyl group’s carbonyl C atom.

(b) The complex after being brought to pH 9 for 1 min and then

rapidly frozen in liquid nitrogen.The various groups in the

structure are represented and colored as in Part a. Note that the

water molecule in Part a has become a hydroxyl substituent

(orange) to the carbonyl C atom, thereby yielding the tetrahedral

intermediate. [Based on X-ray structures by Christopher

Schofield and Janos Hajdu, University of Oxford, U.K. PDBids

(a) 1HAX and (b) 1HAZ.]

(a)

(b)

JWCL281_c15_506-556.qxd 6/5/10 9:10 AM Page 535

from Ser 195, thereby converting its weakly nucleophilic

¬CH

OH group to a highly nucleophilic alkoxide ion,

¬CH



In the process, the anionic charge of Asp 102 was thought

to be transferred, via a tautomeric shift of His 57, to Ser

195. The catalytic triad was therefore originally named the

charge relay system. It is now realized,however,that such a

mechanism is implausible because an alkoxide ion (pK 

15) has far greater proton affinity than does His 57 (pK ⬇

7, as measured by NMR techniques). How, then, can Asp

102 nucleophilically activate Ser 195?

A possible solution to this conundrum has been

pointed out by W.W. Cleland and Maurice Kreevoy and,

independently, by John Gerlt and Paul Gassman. Proton

transfers between hydrogen bonded groups

only occur at physiologically reasonable rates when the

pK of the proton donor is no more than 2 or 3 pH units

greater than that of the protonated form of the proton

acceptor (the height of the kinetic barrier, G

‡

, for the

protonation of an acceptor by a more basic donor in-

creases with the difference between the pK’s of the

donor and acceptor). However, when the pK’s of the hy-

drogen bonding donor (D) and acceptor (A) groups are

nearly equal, the distinction between them breaks down:

The hydrogen atom becomes more or less equally shared

between them . Such low-barrier hydrogen

bonds (LBHBs) are unusually short and strong (they are

also known as short, strong hydrogen bonds): They have,

as studies of model compounds in the gas phase indicate,

association free energies as high as 40 to 80

(D¬H

Asp

102

His

–

Ser

195

Asp

102

His

Ser

195

–

"Charge relay system"

kJ ⴢ mol

1

versus the 12 to 30 kJ ⴢ mol

1

for normal

hydrogen bonds (the energy of the normally covalent

bond is subsumed into the low-barrier hydrogen

bonding system) and a length of 2.55 Å for

and 2.65 Å for versus 2.8 to 3.1 Å

for normal hydrogen bonds.

LBHBs are unlikely to exist in dilute aqueous solution

because water molecules, which are excellent hydrogen

bonding donors and acceptors, effectively compete with

and A for hydrogen bonding sites. However, LB-

HBs may exist in nonaqueous solution and in the active

sites of enzymes that exclude bulk solvent water. If so, an

effective enzymatic “strategy” would be to convert a

weak hydrogen bond in the Michaelis complex to a

strong hydrogen bond in the transition state, thereby fa-

cilitating proton transfer while applying the difference in

the free energy between the normal and low-barrier hy-

drogen bonds to preferentially binding the transition

state. In fact, as Perry Frey has shown, the NMR spec-

trum of the proton linking His 57 to Asp 102 in chy-

motrypsin (which exhibits a particularly large downfield

chemical shift indicative of deshielding) is consistent

with the formation of an LBHB in the transition state

(Fig. 15-25b; the pK’s of protonated His 57 and Asp 102

are nearly equal in the anhydrous environment of the ac-

tive site complex). This presumably promotes proton

transfer from Ser 195 to His 57 as in the charge relay

mechanism. Moreover, an ultrahigh (0.78 Å) resolution

X-ray structure of Bacillus lentus subtilisin by Richard

Bott reveals that the hydrogen bond between His 64 and

Asp 32 of its catalytic triad has an unusually short

distance of 2.62  0.01 Å and that its H atom is nearly

centered between the N and O atoms (note that this

highly accurate protein X-ray structure is one of the very

few in which H atoms are observed and in which short

distances are confidently measured).

Although several studies, such as the foregoing, have re-

vealed the existence of unusually short hydrogen bonds in

enzyme active sites, it is far more difficult to demonstrate

experimentally that they are unusually strong, as LBHBs

are predicted to be. In fact, several studies of the strengths

of unusually short hydrogen bonds in organic model

compounds in nonaqueous solutions suggest that these

hydrogen bonds are not unusually strong. Consequently, a

lively debate has ensued as to the catalytic significance of

LBHBs. Yet if enzymes do not form LBHBs, it remains to

be explained how, in numerous widely accepted enzymatic

mechanisms that we shall encounter, the conjugate base of

an acidic group can abstract a proton from a far more basic

group.

e. Much of a Serine Protease’s Catalytic Activity

Arises from Preferential Transition State Binding

Despite the foregoing, blocking the action of the

catalytic triad through the specific methylation of His 57

by treating chymotrypsin with methyl-p-nitrobenzene

sulfonate

D¬H

N¬H

OO¬H

D¬H

536 Chapter 15. Enzymatic Catalysis

JWCL281_c15_506-556.qxd 6/5/10 9:10 AM Page 536

yields an enzyme that is a reasonably good catalyst: It en-

hances the rate of proteolysis by as much as a factor of 2 

over the uncatalyzed reaction, whereas the native en-

zyme has a rate enhancement factor of ⬃10

. Similarly, the

mutation of Ser 195, His 57, or even all three residues of the

catalytic triad yields enzymes that enhance proteolysis

rates by ⬃5  10

-fold over that of the uncatalyzed reac-

tion. Evidently, the catalytic triad provides a nucleophile

and is an alternate source and sink of protons (general

acid–base catalysis). However, a large portion of chy-

motrypsin’s rate enhancement must be attributed to its pref-

erential binding of the catalyzed reaction’s transition state.

f. Enzymes Have Free Energy Landscapes That

Facilitate Catalysis

How does an enzyme-catalyzed reaction reach its tran-

sition state? Recall that, under physiological conditions,

proteins are highly dynamic entities with structural fluc-

tuations having periods ranging from ⬃10

15

s for bond

vibrations to 1 s or more for triggered conformational

changes (Section 9-4). The turnover times for enzymatic

reactions, 1/k

cat

, are mostly in the range 1 s to 1 s (Table

14-1), but yet the lifetime of a transition state is only

around that of a bond vibration (Section 14-1Cb). Thus,

even for a reaction with a turnover time as little as 1 s,

every atom in the enzyme–substrate complex undergoes

approximately 10

6

/10

15

 10

vibrational excursions be-

tween turnovers. Apparently, the transition state is an

arrangement of substrate and catalytic groups that occurs

extremely rarely through the fluctuations of its component

atoms.

Proteins, as we have seen, are designed by evolution to

fold to their native states via a series of conformational

adjustments that follow funnel-shaped free energy land-

scapes (Section 9-1Ch). A variety of structural, muta-

tional, and theoretical studies indicate that enzyme–sub-

His

–

Methyl-p-nitrobenzene

sulfonate

strate complexes are similarly evolutionarily designed to

structurally rearrange themselves through a progression of

conformational changes that lead to the formation of the

transition state. This explains, for example, why the muta-

tion of a residue far from the active site of an enzyme that

does not appear to have a structurally important role, may

nevertheless significantly reduce the rate of the reaction

that the enzyme catalyzes. Such a mutation perturbs ex-

tended hydrogen bonding networks and long range elec-

trostatic interactions in a way that alters the entire en-

zyme’s spectrum of thermal motions. This changes the

enzyme’s free energy landscape so as to reduce the prob-

ability that it will achieve the transition state in a given

time period.

E. Zymogens

Most proteolytic enzymes are biosynthesized as somewhat

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

these enzymes were synthesized in their active forms, they

would digest the tissues that synthesized them. Indeed,

acute pancreatitis, a painful and sometimes fatal condition

that can be precipitated by pancreatic trauma, is character-

ized by the premature activation of the digestive enzymes

synthesized by this gland.

a. Serine Proteases Are Autocatalytically Activated

Trypsin, chymotrypsin, and elastase are activated ac-

cording to the following pathways:

Trypsin. The activation of trypsinogen, the zymogen of

trypsin, occurs as a two-stage process when trypsinogen en-

ters the duodenum from the pancreas. Enteropeptidase, a

single-pass transmembrane serine protease that is located

in the duodenal mucosa, specifically hydrolyzes trypsino-

gen’s Lys 15¬Ile 16 peptide bond, thereby excising its N-

terminal hexapeptide (Fig. 15-27). This yields the active en-

zyme, which has Ile 16 at its N-terminus. Since this

Section 15-3. Serine Proteases 537

Figure 15-27 Activation of trypsinogen to form trypsin.

Proteolytic excision of the N-terminal hexapeptide is catalyzed

by either enteropeptidase or trypsin.The chymotrypsinogen

residue numbering is used here; that is, Val 10 is actually

trypsinogen’s N-terminus and Ile 16 is trypsin’s N-terminus.

(Asp)

Lys

Trypsinogen

Val

10 15

Ile

. . .

enteropeptidase or

trypsin

Val

(Asp)

Lys

Val

Ile

. . .

Val

Trypsin

JWCL281_c15_506-556.qxd 6/5/10 9:10 AM Page 537

activating cleavage occurs at a trypsin-sensitive site (recall

that trypsin cleaves after Arg and Lys residues), the small

amount of trypsin produced by enteropeptidase also cat-

alyzes trypsinogen activation, generating more trypsin, etc.;

that is, trypsinogen activation is autocatalytic.

Chymotrypsin. Chymotrypsinogen is activated by the spe-

cific tryptic cleavage of its Arg 15¬Ile 16 peptide bond to

form ␲-chymotrypsin (Fig. 15-28). -Chymotrypsin subse-

quently undergoes autolysis (self-digestion) to specifically

excise two dipeptides, Ser 14–Arg 15 and Thr 147–Asn 148,

thereby yielding the equally active enzyme ␣-chymotrypsin

(heretofore and hereafter referred to as chymotrypsin).

The biochemical significance of this latter process, if any, is

unknown.

Elastase. Proelastase, the zymogen of elastase, is activated

similarly to trypsinogen by a single tryptic cleavage that ex-

cises a short N-terminal polypeptide.

b. Biochemical “Strategies” That Prevent Premature

Zymogen Activation

Trypsin activates pancreatic procarboxypeptidases A

and B and prophospholipase A

(the action of phospholi-

pase A

is outlined in Section 25-1) as well as the pancreatic

serine proteases. Premature trypsin activation can conse-

quently trigger a series of events that lead to pancreatic

self-digestion. Nature has therefore evolved an elaborate

defense against such inappropriate trypsin activation. We

have already seen (Section 15-3Da) that pancreatic trypsin

inhibitor binds essentially irreversibly to any trypsin

formed in the pancreas so as to inactivate it. Furthermore,

the trypsin-catalyzed activation of trypsinogen (Fig. 15-27)

occurs quite slowly, presumably because the unusually

large negative charge of its highly evolutionarily conserved

N-terminal hexapeptide repels the Asp at the back of

trypsin’s specificity pocket (Fig. 15-21). Finally, pancreatic

zymogens are stored in intracellular vesicles called zymo-

gen granules whose membranous walls are thought to be

resistant to enzymatic degradation.

c. Zymogens Have Distorted Active Sites

Since the zymogens of trypsin, chymotrypsin, and elastase

have all their catalytic residues, why aren’t they enzymati-

cally active? Comparisons of the X-ray structures of

trypsinogen with that of trypsin and of chymotrypsinogen

with that of chymotrypsin show that on activation, the newly

liberated N-terminal Ile 16 residue moves from the surface of

the protein to an internal position, where its free cationic

amino group forms an ion pair with the invariant anionic Asp

194 (Fig. 15-20). Aside from this change, however, the struc-

tures of these zymogens closely resemble those of their cor-

responding active enzymes. Surprisingly, this resemblance in-

cludes their catalytic triads, an observation which led to the

discovery that these zymogens are actually catalytically ac-

tive, albeit at a very low level. Careful comparisons of the cor-

responding enzyme and zymogen structures revealed the

reason for this low activity: The zymogens’ specificity pockets

and oxyanion holes are improperly formed such that, for ex-

ample,the amide NH of chymotrypsinogen’s Gly 193 points in

the wrong direction to form a hydrogen bond with the tetrahe-

dral intermediate (see Fig. 15-25). Hence, the zymogens’ very

low enzymatic activity arises from their reduced ability to

bind substrate productively and to stabilize the tetrahedral

intermediate. These observations provide further structural

evidence favoring the role of preferred transition state bind-

ing in the catalytic mechanism of serine proteases.

538 Chapter 15. Enzymatic Catalysis

Cys

Chymotrypsinogen

(inactive)

π-Chymotrypsin

(active)

122

136

201

245

Cys

15 16

122

136

201

245

α-Chymotrypsin

(active)

122

136

146 149

201

245

trypsin

chymotrypsin

14 15 + 147 148

Ser Arg Thr Asn

Arg Ile

Leu

Ile

Tyr Ala

Figure 15-28 Activation of chymotrypsinogen by proteolytic cleavage. Both - and -chymotrypsin

are enzymatically active.

See Kinemage Exercise 10-4

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4 DRUG DESIGN

The improvements in medical care over the past several

decades are, in large measure, attributable to the develop-

ment of a huge variety of drugs, which have eliminated or

greatly relieved numerous human ailments. Such medica-

tions include antibiotics (which have enormously reduced

the impact of infectious diseases), anti-inflammatory

agents (which reduce the effects of inflammatory diseases

such as arthritis), analgesics and anesthetics (which make

modern surgical techniques possible), agents that reduce

the incidence and severity of cardiovascular disease and

stroke, antidepressants, antipsychotics, agents that inhibit

stomach acid secretion (which prevent stomach ulcers and

heartburn), agents to combat allergies and asthma, im-

munosuppressants (which make organ transplants possi-

ble), agents used for cancer chemotherapy, and a great va-

riety of other substances.

Early human cultures almost certainly recognized both

the beneficial and toxic effects of indigenous plant and an-

imal products and used many of them as “medications.”

Unfortunately, most of these substances were useless or

even harmful.Although there were sporadic attempts over

the 2500 years preceding the modern era to formulate ra-

tional systems of drug discovery, they had little success be-

cause they were based mainly on unfounded theories and

superstition (e.g., the doctrine of signatures stated that if a

plant resembles a particular body part, it must be designed

by nature to influence that body part) rather than observa-

tion and experiment. Consequently, at the beginning of the

20th century, only three known drugs, apart from folk med-

icines, were effective in treating specific diseases: (1) Digi-

talis, a heart stimulant extracted from the foxglove plant

(Section 20-3Af), was used to treat various heart condi-

tions; (2) quinine (Section 26-4Ak), 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 until several

decades later that the rise of the scientific method coupled

to the rapidly increasing knowledge of physiology, bio-

chemistry, and chemistry led to effective methods of drug

discovery. In fact, the vast majority of drugs in use today

were discovered and developed in the past four decades.

In this section we discuss the elements of drug discovery

and pharmacology (Greek: pharmacon, drug; the science of

drugs, including their composition, uses, and effects).The sec-

tion ends with a consideration of one of the major successes

of modern drug discovery methods, HIV protease inhibitors.

A. Techniques of Drug Discovery

Most drugs act by modifying the function of a particular re-

ceptor in the body or in an invading pathogen. In most

cases, the receptor is a protein to which the drug specifically

binds. It may be an enzyme, a transmembrane channel that

transports a specific substance into or out of a cell (Chapter

20), and/or a protein that participates in an inter- or intra-

cellular signaling pathway (Chapter 19). In all of these

cases, a substance that in binding to a receptor modulates its

function is known as an agonist, whereas a substance that

binds to a receptor without affecting its function but blocks

the binding of agonists is called an antagonist. The bio-

chemical and physiological effects of a drug and its mecha-

nism of action are referred to as its pharmacodynamics.

a. Drug Discovery Is a Complex Procedure

How are new drugs discovered? Nearly all drugs that

have been in use for over 15 years were discovered by

screening large numbers of synthetic compounds and natu-

ral products for the desired effect. Drug candidates that are

natural products are usually discovered by the fractionation

of the organisms in which they occur,which are often plants

used in folk remedies of the conditions of interest. Humans

having the condition whose treatment is being sought can-

not be used as “guinea pigs” in this initial screening process,

and even guinea pigs or other laboratory animals such as

mice or dogs (if they can be made to be suitable models of

the condition under consideration) are too expensive to use

on the many thousands of compounds that are usually

tested. Thus, in vitro screens are initially used, such as the

degree of binding of a drug candidate to an enzyme that is

implicated in a disease of interest, toxicity toward the target

bacteria in the search for a new antibiotic, or effects on a

line of cultured mammalian cells. However, as the number

of drug candidates is winnowed down, more sensitive

screens such as testing in animals are employed.

A drug candidate that exhibits a desired effect is called a

lead compound (or,colloquially, a lead; pronounced leed).A

good lead compound binds to its target receptor with a dis-

sociation constant, K

 1 M. Such a high affinity is neces-

sary to minimize a drug’s less specific binding to other

macromolecules in the body and to ensure that only low

doses of the drug need be taken. For enzyme inhibitors, the

dissociation constant is the inhibitor’s K

or K¿

(Section 14-3).

Other common measures of the effect of a drug are the

, the inhibitor concentration at which an enzyme ex-

hibits 50% of its maximal activity; the ED

, the effective

dose of a drug required to produce a therapeutic effect in

50% of a test sample;the TD

, the mean toxic dose required

to produce a particular toxic effect in animals; and the LD

the mean lethal dose required to kill 50% of a test sample.

For an inhibitor of an enzyme that follows

Michaelis–Menten kinetics, the IC

is determined by meas-

uring the ratio v

for several values of [I] at constant [S],

where v

is the initial velocity of the enzyme when the in-

hibitor concentration is [I]. By dividing Eq. [14.24] by Eq.

[14.38] with ␣ defined according to Eq. [14.37], we see that

[15.13]

When v

 0.5 (50% inhibition),

[15.14]

Consequently, if the measurements of v

are made with

[S]  K

, then [IC

]  K

[I]  [IC

]  K

a1 

[S]



 [S]

a  [S]



 [S]

a1 

[I]

b [S]

Section 15-4. Drug Design 539

JWCL281_c15_506-556.qxd 2/19/10 9:28 PM Page 539

The ratio TD

/ED

is defined as a drug’s therapeutic

index, the ratio of the dose of the drug that produces toxi-

city to that which produces the desired effect. It is, of

course, preferable that a drug have a high therapeutic in-

dex, but this is not always possible.

b. Cathepsin K Is a Drug Target for Osteoporosis

The development of genomic sequencing techniques

(Section 7-2B) and hence the characterization of tens of

thousands of previously unknown genes is providing an

enormous number of potential drug targets. For example,

osteoporosis (Greek: osteon, bone  poros, porous), a con-

dition that afflicts mostly postmenopausal women, is char-

acterized by the progressive loss of bone mass leading to a

greatly increased frequency of bone fracture, most often of

the hip, spine, and wrist. Bones consist of a protein matrix

that is 90% type I collagen (Section 8-2B), in which

spindle- or plate-shaped crystals of hydroxyapatite,

(PO

)

OH, are embedded. Bones are by no means

static structures. They undergo continuous remodeling

through the countervailing action of two types of bone

cells: osteoblasts (Greek:blast, germ cell),which synthesize

bone’s protein matrix in which its mineral component is

laid down; and osteoclasts (Greek: clast, broken), which

solubilize mineralized bone matrix through the secretion

of proteolytic enzymes into an extracellular bone resorp-

tion pit, which is maintained at pH 4.5. The acidic solution

dissolves the bone’s mineral component, thereby exposing

its protein matrix to proteolytic degradation. Osteoporosis

arises when bone resorption outstrips bone formation.

In the search for a drug target for osteoporosis, a cDNA

library (Sections 5-5E and 5-5Fa) was prepared from an

osteoclastoma (a cancer derived from osteoclasts; normally

osteoclasts are very rare cells). Around 4% of these cDNAs

encode a heretofore unknown protease, which was named

cathepsin K (cathepsins are proteases that occur in the lyso-

some). Further studies, both at the cDNA and protein levels,

indicated that cathepsin K is only expressed at high levels in

osteoclasts. Microscopic examination of osteoclasts that had

been stained with antibodies directed against cathepsin K

revealed that this enzyme is localized at the contact site be-

tween osteoclasts and the bone resorption pit. Subsequently,

it was shown that mutations in the gene encoding cathepsin

K are the cause of pycnodysostosis, a rare hereditary disease

which is characterized by hardened and fragile bones, short

stature, skull deformities, and osteoclasts that demineralize

bone normally but do not degrade its protein matrix. Evi-

dently, cathepsin K functions to degrade the protein matrix

of bone and hence is an attractive drug target for the treat-

ment of osteoporosis. Indeed, several cathepsin K inhibitors

are in clinical trials (Section 15-4Bb).

c. SARs and QSARs Are Useful Tools for

Drug Discovery

A lead compound is used as a point of departure to design

more efficacious compounds. Experience has shown that

even minor modifications to a drug candidate can result in

major changes in its pharmacological properties. Thus, one

might place methyl, chloro, hydroxyl, or benzyl groups at

various places on a lead compound in an effort to improve

its pharmacodynamics. For most drugs in use today, 5 to 10

thousand related compounds were typically synthesized in

generating the medicinally useful drug.These were not ran-

dom procedures but were guided by experience as medici-

nal chemists tested various derivatives of a lead compound:

For those compounds that had improved efficacy, deriva-

tives were made and tested; etc. This process has been sys-

tematized through the use of structure–activity relation-

ships (SARs): the determination, via synthesis and

screening, of which groups on a lead compound are impor-

tant for its drug function and which are not. For example, if

a phenyl group on a lead compound interacts hydrophobi-

cally with a flat region of its receptor, then hydrogenating

the phenyl ring to form a nonplanar cyclohexane ring will

yield a compound with reduced affinity for the receptor.

A logical extension of the SAR concept is to quantify it,

that is, to determine a quantitative structure–activity rela-

tionship (QSAR). This idea is based on the premise that

there is a relatively simple mathematical relationship be-

tween the biological activity of a drug and its physicochem-

ical properties. For instance, if the hydrophobicity of a drug

is important for its biological activity, then changing the

substituents on the drug so as to alter its hydrophobicity

will affect its activity. A measure of the substance’s hy-

drophobicity is its partition coefficient, P, between the two

immiscible solvents, octanol and water, at equilibrium:

[15.15]

Biological activity may be expressed as 1/C, where C is the

drug concentration required to achieve a specified level of

biological function (e.g., IC

).Then a plot of log 1/C versus

log P (the use of logarithms keeps the plot on a manage-

able scale) for a series of derivatives of the lead compound

having a relatively small range of log P values often indi-

cates a linear relationship (Fig. 15-29a), which can there-

fore be expressed:

[15.16]

Here k

and k

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-

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-

scribed by a quadratic equation:

[15.17]

Of course, the biological activities of few substances de-

pend only on their hydrophobicities. A QSAR can there-

fore simultaneously take into account several physico-

chemical properties of substituents such as their pK values,

van der Waals radii, hydrogen bonding energy, and confor-

mation.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 biologi-

cal activity of a lead compound has proven to be a valuable

tool in drug discovery.

log

b k

(log P)

 k

log P  k

log a

b k

log P  k

P 

concentration of drug in octanol

concentration of drug in water

540 Chapter 15. Enzymatic Catalysis

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