Chymotrypsin Is Activated by Proteolysis

1
Chymotrypsin Is Activated by Proteolysis
Chymotrypsinogen (inactive)
245
Trypsin
p-Chymotrypsin (active)
R15-I16
p-Chymotrypsin
S14-R15
T147-N148
Adapted from Campbell (1999) Biochemistry (3d)
p.179
I16
L13
A149
Y146
Disulfide bonds
a-Chymotrypsinogen (active)
2
Charge Relay in Active Site
Ser 195
Asp 102
His 57
Active Ser
Adapted from Alberts et al (2002) Molecular
Biology of the Cell (4e) p.158
3
pH Influences Chymotrypsin Activity
Relative Activity
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.162
4
pH Influences Net Charge of Protein
Buffer pH
Isoelectric point, pI

Juang RH (2004) BCbasics
-
0

Net Charge of a Protein
5
Imidazole on Histidine Is Affected by pH
pH 6
pH 7

Adapted from Dressler Potter (2000) Discovering
Enzymes, p.163
Inactive
Adapted from Alberts et al (2002) Molecular
Biology of the Cell (4e) p.158
6
Chymotrypsin Produces New Ile16 N-Terminal
New NH2-terminus
pKa
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.165
7
New Ile16 N-Terminal Stabilizes Asp194
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.206
Catalytic Triad
Gly 193
His 57
Ser 195
Asp 194
Asp 102
NH3
Ile 16
Nelson Cox (2000) Lehninger Principles of
Biochemistry (3e) p.112
8
Chymotrypsin Ser195 Inhibited by DIFP
Diisopropyl-fluorophosphate (DIFP)
X
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.167
9
Addition of Substrate Blocks DIFP Inhibition
No substrate
DIFP
X
Add substrate
DIFP substrate
S
Reaction time
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.167
10
Chymotrypsin Also Catalyzes Acetate
Hartley Kilby
Nitrophenol acetate
Chymotrypsin
H2O
Peptide bond
Acetate
Nitrophenol
Ester bond
No acetate was detected at early stage
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.168
11
Two-Stage Catalysis of Chymotrypsin
Acylation
Nitrophenol acetate
Kinetics of reaction
Deacylation (slow step)
CH3COOH
H2O
Two-phase reaction
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.169
12
Extra Negative Charge Was Neutralized
E S
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.179
13
Active Site Stabilizes Transition State
Catalytic Triad
Active Site
Adapted from Dressler Potter (1991) Discovering
Enzymes, p.197
Specificity Site