Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

FIGURE 16.26

O

R C N R'

H

O

R C N R'

H

+

+

O

O–

R

C

+

E

C

O

R'

NH2

O

E

C

O

Acyl-enzyme

intermediate

O–

H

+

COO–

E

C

E

C

N

R'

R

O

O

Amino-enzyme

intermediate

16.12 ● The Aspartic Proteases

521

● Acyl-enzyme and aminoenzyme intermediates originally proposed for aspartic proteases were modeled after the acylenzyme intermediate of the serine proteases.

the aspartate groups in the free enzyme or in the enzyme-substrate complex. Thus, in pepsin, for example, Asp32 may be deprotonated while Asp215 is protonated, or vice versa.

In the most widely accepted mechanism (Figure 16.27), substrate binding is followed by a step in which two concerted proton transfers facilitate nucleophilic attack on the carbonyl carbon of the substrate by water. In the mechanism shown, Asp32 acts as a general base, accepting a proton from an activesite water molecule, whereas Asp215 acts a general acid, donating a proton to the oxygen of the peptide carbonyl group. By virtue of these two proton transfers, nucleophilic attack occurs without explicit formation of hydroxide ion at the active site.

The resulting intermediate is termed an amide dihydrate. Note that the protonation states of the two aspartate residues are now opposite to those in the free enzyme (Figure 16.27).

Breakdown of the amide dihydrate occurs by a mechanism similar to its formation. The ionized aspartate carboxyl (Asp32 in Figure 16.27) acts as a general base to accept a proton from one of the hydroxyl groups of the amide dihydrate, while the protonated carboxyl of the other asparate (Asp215 in this case) simultaneously acts as a general acid to donate a proton to the nitrogen atom of one of the departing peptide products.

	Asp32
	C			O H
			–				H
	O
					..
	...		.....		O
					O
	H
					C		R
					NHR'
	O

Asp32
C			O
O
			HO	OH
H			C
...
			NHR'	R
O

	Asp32
C					O
			–
O
... H						O
H						C
		......
O					..NHR'

OH

R

					–
C			O	C			O	C			O
	Asp215				Asp215				Asp215
			(a)				(b)				(c)

FIGURE 16.27 ● A mechanism for the aspartic proteases. In the first step, two concerted proton transfers facilitate nucleophilic attack of water on the substrate carbonyl carbon. In the third step, one aspartate residue (Asp32 in pepsin) accepts a proton from one of the hydroxyl groups of the amine dihydrate, and the other aspartate (Asp215) donates a proton to the nitrogen of the departing amine.

Asp32

C O

O O OH

C

H

O				R
	–			NH2R'
C			O
	Asp215
				(d)

522 Chapter 16 ● Mechanisms of Enzyme Action

The AIDS Virus HIV-1 Protease Is an Aspartic Protease

Recent research on acquired immune deficiency syndrome (AIDS) and its causative viral agent, the human immunodeficiency virus (HIV-1), has brought a new aspartic protease to light. HIV-1 protease cleaves the polyprotein products of the HIV-1 genome, producing several proteins necessary for viral growth and cellular infection. HIV-1 protease cleaves several different peptide linkages in the HIV-1 polyproteins, including those shown in Figure 16.28. For example, the protease cleaves between the Tyr and Pro residues of the sequence Ser- Gln-Asn-Tyr-Pro-Ile-Val, which joins the p17 and p24 HIV-1 proteins.

The HIV-1 protease is a remarkable viral imitation of mammalian aspartic proteases: It is a dimer of identical subunits that mimics the two-lobed monomeric structure of pepsin and other aspartic proteases. The HIV-1 protease subunits are 99-residue polypeptides that are homologous with the individual domains of the monomeric proteases. Structures determined by X-ray diffraction studies reveal that the active site of HIV-1 protease is formed at the interface of the homodimer and consists of two aspartate residues, designated Asp25 and Asp25 , one contributed by each subunit (Figure 16.29). In the homodimer, the active site is covered by two identical “flaps,” one from each subunit, in contrast to the monomeric aspartic proteases, which possess only a single active-site flap.

Enzyme kinetic measurements by Thomas Meek and his collaborators at SmithKline Beecham Pharmaceuticals have shown that the mechanism of HIV-1 protease is very similar to those of other aspartic proteases (Figure 16.30). Two concerted proton transfers by the aspartate carboxyls facilitate nucleophilic attack by water on the carbonyl carbon of the peptide substrate. If the protease-substrate complex is incubated with H218O, incorporation of 18O into the peptide carbonyl group can be measured. Thus, not only is the formation of the amide dihydrate reversible, and the two hydroxyl groups of the dihydrate equivalent, but the protonation states of the active-site carboxyl groups must interchange (Figure 16.30). The simplest model would involve direct proton exchange across the Asp-Asp dyad as shown. The symmetrical nature of the active-site aspartyl groups is consistent with the idea of facile exchange of the proton between the two Asp residues.

The observation that 18O can accumulate in the substrate from H218O also implies that formation and reversal of the amide dihydrate must be faster than

FIGURE 16.28 ● HIV mRNA provides the genetic information for synthesis of a polyprotein. Proteolytic cleavage of this polyprotein by HIV protease produces the individual proteins required for viral growth and cellular infection.

gag		pol
mRNA
	Translation
Protein		(gag–pol polyprotein)
	Protease
p17	p11(protease)
Proteins
p24	p66/51 (reverse transcriptase)
	p15	p32(integrase)

p7 p6

(Adapted

FIGURE 16.30

FIGURE 16.29

16.12 ● The Aspartic Proteases

523

● (left) HIV-1 protease complexed with the inhibitor Crixivan® (red) made by Merck. The flaps (residues 46–55 from each subunit) covering the active site are shown in green and the active site aspartate residues involved in catalysis are shown in white.

(right) The close-up of the active site shows the interaction of Crixivan® with the carboxyl groups of the essential aspartate residues.

its breakdown to form product. Kinetic studies by Meek and his coworkers are consistent with a model in which breakdown of the dihydrate intermediate is the rate-determining step for the protease reaction. These studies also show that the transition state of this step involves two proton transfers—with one aspartate acting as a general acid to donate a proton to the departing proline and the other aspartate acting as a general base to abstract a proton from one of the hydroxyl groups, facilitating collapse of the dihydrate to a carboxyl group. Note that the events of the breakdown to form product are simply the reversal of the steps that formed the intermediate.

HO				HO
H	H				H	H
	N		N			N	O		N
		O						O		R'
	H					H
	H			R'		H			H	O
		H		O
O		O				O –
		H								O
				O				H
		O	–						O
				O			O
Asp25										Asp25'
						Asp25
				Asp25'
								Amide
								dihydrate
HO				HO
H	H				H	H
	N		N			N	O		N
		O						O		R'
	H					H
		H					H
				R'		H			H	O
O –		O		O
		H		O		O			–	O
	O		O
		H					O		O
Asp25				Asp25'
						Asp25			Asp25'

● A mechanism for the incorporation of 18O from H218O into peptide substrates in the HIV protease reaction.

from Hyland, L., et al., 1991. Biochemistry 30:8441– 8453.)

HO

H	H
	N	O	N
			R'
	H	O
		H
H+			H O
	O
		H	O
			–
		O	O
	Asp25		Asp25'

524 Chapter 16 ● Mechanisms of Enzyme Action

H U M A N B I O C H E M I S T R Y

Protease Inhibitors Give Life to AIDS Patients

Infection by HIV was once considered a death sentence, but the emergence of a new family of drugs called protease inhibitors has made it possible for some AIDS patients to improve their overall health and extend their lives. These drugs are all specific inhibitors of the HIV protease. By inhibiting the protease, they prevent the development of new virus particles in the cells of infected patients. Clinical testing has shown that a combination of drugs—including a protease inhibitor together with a reverse transcriptase inhibitor like AZT—can reduce the human immunodeficiency virus (HIV) to undetectable levels in about 40 to 50% of infected individuals. Patients who respond successfully to this combination therapy have experienced dramatic improvement in their overall health and a substantially lengthened lifespan.

Four of the protease inhibitors approved for use in humans by the U.S. Food and Drug Administration are shown below: Crixivan® by Merck, Invirase® by Hoffman-LaRoche, Norvir® by Abbott, and Viracept® by Agouron. These drugs were all developed from a “structure-based” design strategy; that is, the drug molecules were designed to bind tightly to the active site of the HIV-1 protease. The backbone OH-group in all these substances inserts between the two active-site carboxyl groups of the protease.

In the development of an effective drug, it is not sufficient merely to show that a candidate compound can cause the desired

biochemical effect. It must also be demonstrated that the drug can be effectively delivered in sufficient quantities to the desired site(s) of action in the organism, and that the drug does not cause undesirable side effects. The HIV-1 protease inhibitors shown here fulfill all of these criteria. Other drug candidates have been found that are even better inhibitors of HIV-1 protease in cell cultures, but many of these fail the test of bioavailability—the ability of a drug to be delivered to the desired site of action in the organism.

Candidate protease inhibitor drugs must be relatively specific for the HIV-1 protease. Many other aspartic proteases exist in the human body and are essential to a variety of body functions, including digestion of food and processing of hormones. An ideal drug thus must strongly inhibit the HIV-1 protease, must be delivered effectively to the lymphocytes where the protease must be blocked, and should not adversely affect the activities of the essential human aspartic proteases.

A final but important consideration is viral mutation. Certain mutant HIV strains are resistant to one or more of the protease inhibitors, and even for patients who respond initially to protease inhibitors it is possible that mutant viral forms may eventually thrive in the infected individual. The search for new and more effective protease inhibitors is ongoing.

	H	O		H
					N	OH		H	OH
N	N	N	N	H		N		N
				N
O		H	OH					O
H2N	C		O	NH	NH	O
	O
Invirase (Saquinavir)						Crixivan (Indinavir)
S		O NH			O	H			O
O			CH3SO3H	N		N			S
OH					N			N
		N		N					O
N
H	OH	H		S	H	O	OH	H	N
		H
Viracept (Nelfinavir mesylate)					Norvir (Ritonavir)

C R I T I C A L D E V E L O P M E N T S I N B I O C H E M I S T R Y

The pH Dependence of Aspartic Proteases and HIV-1 Protease

The first hint that two active-site carboxyl groups—one protonated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right.

If activity increases dramatically as pH is increased, catalysis may depend on a deprotonated group that may normally act as a general base, accepting a proton from the substrate or a water molecule, for example (a). Protonation of this group at lower pH prevents it from accepting another proton (from the substrate or water, for example).

On the other hand, if activity decreases sharply as pH is raised, activity may depend on a protonated group, which may act as a general acid, donating a proton to the substrate or a catalytic water molecule (b). At high pH, the proton dissociates and is not available in the catalytic events.

The bell-shaped curve in part (c) of the figure combines both of these behaviors, so that activity first increases, then decreases, as pH is increased. This is consistent with the involvement of two ionizable groups—one with a low pK a that acts as a base above its pK a, and a second group with a higher pK a that acts as an acid below its pK a.

Kinetic studies with pepsin have produced bell-shaped curves for a variety of substrate peptides; see below, (a).

As such data are fitted to calculated curves, dissociation constants can be estimated for the ionizable groups at the active site. In pepsin, the general base exhibits a pK a of approximately 1.4, whereas the general acid displays a pK a of about 4.3. Compare these values with the expected pK a for an aspartate carboxyl group in a protein of 4.2 to 4.6. The value for the aspartyl group acting as a general acid in pepsin is typical, but that for the aspartyl group acting as a general base is unusually low.

The pH dependence of HIV-1 protease has been assessed by measuring the apparent inhibition constant for a synthetic substrate analog (b). The data are consistent with the catalytic involvement of ionizable groups with pK a values of 3.3 and 5.3. Maximal enzymatic activity occurs in the pH range between these two values. On the basis of the accumulated kinetic and structural data on HIV-1 protease, these pK a values have been ascribed to the

(a)				B + H+
Enzyme activity
		B+		H
				pH
(b)
activity		A		H
Enzyme				A– + H+

pH

(c)

Enzyme activity

B+

H

B + H+

AH

A– + H+

pH

Bell-shaped activity versus pH profiles arise from two separate active-site ionizations. (a) Enzyme activity increases upon deprotonation of B -H.

(b) Enzyme activity decreases upon deprotonation of A-H. (c) Enzyme activity is maximal in the pH range where one ionizable group is deprotonated (as B:) and the other group is protonated (as A-H).

two active-site aspartate carboxyl groups. Note that the value of 3.3 is somewhat low for an aspartate side chain in a protein, whereas the value of 5.3 is somewhat high.

(a)			(b)
Enzyme activity		Pepsin	Inhibition constants		HIV	Protease

pH-rate profiles for (a) pepsin and (b) HIV protease. (Adapted from Denburg, J., et al., 1968. Journal of the American Chemical Society

90:479–486, and Hyland, J., et al., 1991. Biochemistry

30:8454–8463.)

0	1	2	3	4	5	6	3	4	5	6	7
			pH						pH

525

526Chapter 16 ● Mechanisms of Enzyme Action

16.13● Lysozyme

Lysozyme is an enzyme that hydrolyzes polysaccharide chains. It ruptures certain bacterial cells by cleaving the polysaccharide chains that make up their cell wall. Lysozyme is found in many body fluids, but the most thoroughly studied form is from hen egg whites. The Russian scientist P. Laschtchenko first described the bacteriolytic properties of hen egg white lysozyme in 1909. In 1922, Alexander Fleming, the London bacteriologist who later discovered penicillin, gave the name lysozyme to the agent in mucus and tears that destroyed certain bacteria, because it was an enzyme that caused bacterial lysis.

As seen in Chapter 9, bacterial cells are surrounded by a rigid, strong wall of peptidoglycan, a copolymer of two sugar units, N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). Both of these sugars are N-acetylated analogs of glucosamine, and in bacterial cell wall polysaccharides, they are joined in(1 n 4) glycosidic linkages (Figure 16.31). Lysozyme hydrolyzes the glycosidic bond between C-1 of NAM and C-4 of NAG, as shown in Figure 16.31, but does not act on the (1 n 4) linkages between NAG and NAM.

Lysozyme is a small globular protein composed of 129 amino acids (14 kD) in a single polypeptide chain. It has eight cysteine residues linked in four disulfide bonds. The structure of this very stable protein was determined by X-ray crystallographic methods in 1965 by David Phillips (Figure 16.32). Although X-ray structures had previously been reported for proteins (hemoglobin and myoglobin), lysozyme was the first enzyme structure to be solved by crystallographic (or any other) methods. Although the location of the active site was not obvious from the X-ray structure of the protein alone, X-ray studies of lysozyme-inhibitor complexes soon revealed the location and nature of the active site. Since it is an enzyme, lysozyme cannot form stable ES complexes for structural studies, because the substrate is rapidly transformed into products. On the other hand, several substrate analogs have proven to be good com-

CH2OH

CH2OH

CH2OH

CH2OH

O

O

O

O

OH

O

OR

O

OH

O

OR

NH

NH

NH

NH

C

O

C

O

C

O

C

O

CH3

CH3

CH3

CH3

NAG

NAM

NAG

NAM

H2O

CH2OH

CH2OH

CH2OH

CH2OH

O

O OH

O

O

OH

O

OR

OH

O

OR

HO

NH

NH

NH

NH

C

O

C

O

C

O

C

O

CH3

CH3

CH3

CH3

NAG

NAM

NAG

NAM

FIGURE 16.31 ● The lysozyme reaction.

petitive inhibitors of lysozyme that can form complexes with the enzyme stable enough to be characterized by X-ray crystallography and other physical techniques. One of the best is a trimer of N-acetylglucosamine, (NAG)3 (Figure 16.33), which is hydrolyzed by lysozyme at a rate only 1/60,000 that of the native substrate (Table 16.4). (NAG)3 binds at the enzyme active site by forming five hydrogen bonds with residues located in one-half of a depression or crevice that spans the surface of the enzyme (Figure 16.34). The few hydrophobic residues that exist on the surface of lysozyme are located in this depression, and they may participate in hydrophobic and van der Waals interactions with (NAG)3, as well as the normal substrate. The absence of charged groups on (NAG)3 precludes the involvement of electrostatic interactions with the enzyme. Comparisons of the X-ray structures of the native lysozyme and the lysozyme-(NAG)3 complex reveal that several amino acid residues at the active site move slightly upon inhibitor binding, including Trp62, which moves about 0.75 Å to form a hydrogen bond with a hydroxymethyl group (Figure 16.35).

Model Studies Reveal a Strain-Induced Destabilization of a Bound Substrate on Lysozyme

One of the premises of lysozyme models is that the native substrate would occupy the rest of the crevice or depression running across the surface of the enzyme, because there is room to fit three more sugar residues into the crevice and because the hexamer (NAG)6 is in fact a good substrate for lysozyme (Table 16.4). The model building studies refer to the six sugar residue-binding subsites in the crevice with the letters A through F, with A, B, and C representing the part of the crevice occupied by the (NAG)3 inhibitor (Figure 16.35). Modeling studies clearly show that NAG residues fit nicely into subsites A, B, C, E, and F of the crevice, but that fitting a residue of the (NAG)6 hexamer into site D requires a substantial distortion of the sugar (out of its preferred chair conformation) to prevent steric crowding and overlap between atoms C-6 and O-6 of the sugar at the D site and Ile98 of the enzyme. This distorted sugar residue is adjacent to the glycosidic bond to be cleaved (between sites D and E), and the inference is made that this distortion or strain brings the substrate closer to the transition state for hydrolysis. This is a good example of strain-induced destabilization of an otherwise favorably binding substrate (Section 16.4). Thus, the overall binding interaction of the rest of the sugar substrate would be favorable ( G 0), but distortion of the ring at the D site uses some of this binding energy to raise the substrate closer to the transition state for hydrolysis, an example of stabilization of a transition state (relative to the simple enzyme-substrate complex). As noted in Section 16.4, distortion is one of the molecular mechanisms that can lead to such transition-state stabilization.

	CH2OH		CH2OH		CH2OH
HO	O	O	O	O	O	OH
	HO		HO		HO
	NH		NH		NH
	C O		C	O	C O
	CH3		CH3		CH3
	NAG		NAG		NAG

FIGURE 16.33 ● (NAG)3, a substrate analog, forms stable complexes with lysozyme.

16.13 ● Lysozyme

527

FIGURE 16.32 ● The structure of lysozyme. Glu35 and Asp52 are shown in white.

Table 16.4

Hydrolysis Rate Constants for Model Oligosaccharides with Lysozyme

Oligosaccharide	Rate Constant, k cat(s 1)
(NAG-NAM)3	0.5
(NAG)6	0.25
(NAG)5	0.033
(NAG)4	7	10 5
(NAG)3	8	10 6
(NAG)2	2.5	10 8

Substrate

FIGURE 16.34 ● The lysozyme-enzyme- substrate complex. (Photo courtesy of John Rupley, University of Arizona)

528 Chapter 16 ● Mechanisms of Enzyme Action

tions the

Lysozyme

Asn44

Trp62

Gln57

Glu35

C

Substrate-binding cleft

O

NH2

N

C

C

CH3 ......

H

CH3

O

O

O

NH2

CH3

O

Asn59

H3C

R

H3C

O

C

R

....

C

O

H

....

C

R

N

....

H

C

O

H

N

O

H

H

O

C

O

H

N

H

N

O

H

N

CH2

O

N

OH

H

O

O

O

O

O

O

.....

......

O

OH

.......

O

CH2

O

O

CH2

N

O

H O

O

H

C

H

.....NAM O

NAG CH2OH

NAM

O

C

H

NAG

.....

NAM

...

O

CH2OH

C

NAG

H2N

NH2

....

H

CH3

.....

NH

O

....

H

H

101

C

NH2

...

O

O

N

Asp

O

Arg114

Asn37

C

Lysozyme

C

C

Ala107

Phe or Trp34

cuts

Gln57

Trp63

FIGURE 16.36

D site

O

C1

O 18 OH

C1

D site

E site

O

4C

O

H218O

HO

+ 4C

O

E site

16.13 ● Lysozyme

529

● The C1–O bond, not the O–C4 bond, is cleaved in the lysozyme reaction. 18O from H218O is thus incorporated at the C1 position.

The Lysozyme Mechanism Involves General Acid–Base Catalysis

The mechanism of the lysozyme reaction is shown in Figures 16.36 and 16.37. Studies using 18O-enriched water showed that the C1OO bond is cleaved on the substrate between the D and E sites. Hydrolysis under these conditions incorporates 18O into the C1 position of the sugar at the D site, not into the oxygen at C4 at the E site (Figure 16.36). Model building studies place the cleaved bond approximately between protein residues Glu35 and Asp52. Glu35 is in a nonpolar or hydrophobic region of the protein, whereas Asp52 is located in a much more polar environment. Glu35 is protonated, but Asp52 is ionized (Figure 16.37). Glu35 may thus act as a general acid, donating a proton to the oxygen atom of the glycosidic bond and accelerating the reaction. Asp52, on the other hand, probably stabilizes the carbonium ion generated at the D site upon bond cleavage. Formation of the carbonium ion may also be enhanced by the strain on the ring at the D site. Following bond cleavage, the product formed at the E site diffuses away, and the carbonium ion intermediate can then react with H2O from the solution. Glu35 can now act as a general base, accepting a proton from the attacking water. The tetramer of NAG thus formed at sites A through D can now be dissociated from the enzyme.

On the basis of the above, the rate acceleration afforded by lysozyme appears to be due to (a) general acid catalysis by Glu35; (b) distortion of the sugar ring at the D site, which may stabilize the carbonium ion (and the transition state); and (c) electrostatic stabilization of the carbonium ion by nearby Asp52. The overall kcat for lysozyme is about 0.5/sec, which is quite slow (Table

Asp32

Asp32

C

O H

C

O

–

H

O

O

..

...

.....

O

O

HO

OH

H

C

R

H

C

...

NHR'

NHR'

R

O

O

–

C

O

C

O

Asp215

Asp215

(a)

(b)

FIGURE 16.37 ● A mechanism for the lysozyme reaction.

	Asp32
C					O
			–
O
... H						O	OH
H						C
		......
O					..NHR'		R

C O

Asp215

(c)

Asp32

C O

O O OH

C

H

O				R
	–			NH2R'
C			O
	Asp215
				(d)

530 Chapter 16 ● Mechanisms of Enzyme Action

14.4) compared with that for other enzymes. On the other hand, the destruction of a bacterial cell wall may only require hydrolysis of a few polysaccharide chains. The high osmotic pressure of the cell ensures that cell rupture will follow rapidly. Thus, lysozyme can accomplish cell lysis without a particularly high kcat.

PROBLEMS

1. Tosyl-L-phenylalanine chloromethyl ketone (TPCK) specifically inhibits chymotrypsin by covalently labeling His57.

	O	CH2	O
CH3	B	A	B
	SONHOCHOCOCH2Cl
	B

O

Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

a.Propose a mechanism for the inactivation reaction, indicating the structure of the product(s).

b.State why this inhibitor is specific for chymotrypsin.

c.Propose a reagent based on the structure of TPCK that might be an effective inhibitor of trypsin.

2. In this chapter, the experiment in which Craik and Rutter replaced Asp102 with Asn in trypsin (reducing activity 10,000-fold) was discussed.

a.On the basis of your knowledge of the catalytic triad structure in trypsin, suggest a structure for the “uncatalytic triad” of Asn- His-Ser in this mutant enzyme.

b.Explain why the structure you have proposed explains the reduced activity of the mutant trypsin.

c.See the original journal articles (Sprang, et al., 1987. Science 237:905–909 and Craik, et al., 1987. Science 237:909–913) to see what Craik and Rutter’s answer to this question was.

3.Pepstatin (see page 531) is an extremely potent inhibitor of the monomeric aspartic proteases, with KI values of less than 1 nM. a. On the basis of the structure of pepstatin, suggest an explanation for the strongly inhibitory properties of this peptide.

b. Would pepstatin be expected to also inhibit the HIV-1 protease? Explain your answer.

4.The kcat for alkaline phosphatase–catalyzed hydrolysis of methylphosphate is approximately 14/sec at pH 8 and 25°C. The rate constant for the uncatalyzed hydrolysis of methylphosphate

under the same conditions is approximately 1 10 15/sec. What is the difference in the free energies of activation of these two reactions?

Chymotrypsinogen (inactive)

1

S

S	S

S

S

S

S

S	S

245

S

π -Chymotrypsin

(active)	S	S

1	15	16

S

S

S

S

S

S	S

245

S

α -Chymotrypsin (active)

1

13 Leu

16 Ile

S

S

	Ser Arg
	14	15
	S
146	149
Tyr	Ala

	Thr Asn
	147 148
S	S	S

245

S