Transcript Chymotrypsin Is Activated by Proteolysis

Chymotrypsin Is Activated by Proteolysis
Chymotrypsinogen (inactive)
245
p-Chymotrypsin
S14-R15
L13
I16
a-Chymotrypsinogen (active)
T147-N148
Y146
A149
Disulfide bonds
Adapted from Campbell (1999) Biochemistry (3d) p.179
p-Chymotrypsin (active)
R15-I16
Trypsin
Asp 102
H–N
C
N
H–O–CH2
C C
H
CH2
Ser
195
His 57
Active Ser
H
O
C–O–H
Asp 102
N
C
N–H
C C
H
CH2
His 57
- O–CH
2
Ser
195
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158
=
O
C–O -
=
Charge Relay in Active Site
H
pH Influences Chymotrypsin Activity
Relative Activity
5
6
7
8
9
10
11
pH
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162
10
9
8
7
Isoelectric point,
pI
+
+
6
5
4
3
0
Net Charge of a Protein
-
Juang RH (2004) BCbasics
pH Influences Net Charge of Protein
Buffer pH
pH 6
H
H–N
C
C
+
N–H
H+
H–N
C
C
H
=
Asp 102
H–N
C
C
N
C
H
H
O
C–O -
pH 7
H
Inactive
+
N–H H–O–CH2
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
6
7
8
I16
Y146
9 10 11
NH2– Ile 16
Asp 194
pH 9
–CH2COO+ NH
3–
pKa
pH 10
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
+NH
3
Ile 16
Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.112
Chymotrypsin Ser195 Inhibited by DIFP
Diisopropyl-fluorophosphate (DIFP)
X
=
O
(CH3)2CH–O– P –O–CH(CH3)2
O
=
F
(CH3)2CH–O– P –O–CH(CH3)2
O-…H
O
CH2
CH2
Ser 195
Ser 195
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
Addition of Substrate Blocks DIFP Inhibition
100
No substrate
Percent Inhibition of activity (%)
+ DIFP
X
50
+ DIFP & substrate
Add substrate
S
0
Reaction time
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
Chymotrypsin Also Catalyzes Acetate
Hartley & Kilby
O
-C NH
Nitrophenol acetate
O
CH3–C–O–
+ H 2O
–NO2
Chymotrypsin
Peptide bond
O
-C OEster bond
HO–
–NO2
Acetate
O
CH3–C–OH
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
CH3–C–O–
–NO2
Acylation
O
CH3–C
–NO2
O
C
O-H
C
+ H2O
Nitrophenol
CH3COOH
Deacylation (slow step)
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