Chymotrypsin Is Activated by Proteolysis

Chymotrypsinogen (inactive)
245

-Chymotrypsin
S14-R15
L13

I16

-Chymotrypsinogen (active)

T147-N148
Y146

A149
Disulfide bonds

Adapted from Campbell (1999) Biochemistry (3d) p.179

-Chymotrypsin (active)
R15-I16

Trypsin

CO

Asp 102

HN

HOCH2

C C
H
CH2

Ser
195

His 57

O
COH

Asp 102

Active Ser

H
N

NH

C C
H
CH2
His 57

OCH2

Ser
195

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158

Charge Relay in Active Site

pH Influences Chymotrypsin Activity

Relative Activity
5

10

11

pH
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162

10
9
8
7
Isoelectric point,
pI

6
5
4
3

Net Charge of a Protein

Juang RH (2004) BCbasics

pH Influences Net Charge of Protein

Buffer pH

pH 6

HN
C

NH

HN

C
H

Asp 102

HN

N
C
H

CO -

pH 7

Inactive

+
NH HOCH2

C C-H
CH2

Ser
195

Adapted from Dressler & Potter (2000) Discovering Enzymes, p.163

Imidazole on Histidine Is Affected by pH

His 57
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158

Chymotrypsin Produces New Ile16 N-Terminal

New NH2-terminus
Relative activity

L13

pH 5

I16

Y146

9 10 11

NH2 Ile 16
Asp 194

CH2COO
+

pH 9

pKa

pH 10

NH3 Ile 16
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165

New Ile16 N-Terminal Stabilizes Asp194

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.206

Catalytic Triad
His 57

Ser 195

Asp 102

Gly 193

Asp 194

NH3
Ile 16

Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.112

Chymotrypsin Ser195 Inhibited by DIFP

Diisopropyl-fluorophosphate (DIFP)

(CH3)2CHO P OCH(CH3)2
O

(CH3)2CHO P OCH(CH3)2

O-H

CH2

CH2

Ser 195

Ser 195
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

Addition of Substrate Blocks DIFP Inhibition

100
Percent Inhibition of activity (%)

+ DIFP

No substrate

X
50
+ DIFP & substrate

Add substrate

Reaction time
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

Chymotrypsin Also Catalyzes Acetate

Hartley & Kilby

O
-C NH
Peptide bond

O
-C OEster bond

Nitrophenol acetate

O
CH3CO
+ H 2O

NO2
Chymotrypsin

HO
Acetate

O
CH3COH

NO2

Nitrophenol

No acetate was detected at early stage

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168

Two-Stage Catalysis of Chymotrypsin

O Nitrophenol acetate
CH3CO
NO2

Acylation

O
CH3C

NO2

O
C

O-H
C

Deacylation (slow step)

+ H2 O

Nitrophenol

CH3COOH

Kinetics of reaction

OC

HO

Two-phase
reaction
Time (sec)

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169

Extra Negative Charge Was Neutralized

-C-C-N-C-C-N-C-C-NH
H
O-C NHO H

O-C NHO H

O
-C NH

E+S

O
-C-OH
NH2Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179

Active Site Stabilizes Transition State

Gly 193

Catalytic Triad
Asp 194

Met 192
His 57

Active Site
Cys 191

Asp 102

Thr 219
Cys 220

Specificity Site

Ser 214
Trp 215

Gly 216

Ser 218
Ser 217

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.197

Ser 195