chymotrypsin is activated by proteolysis adapted from campbell (1999) biochemistry (3d) p.179 245...
TRANSCRIPT
Chymotrypsin Is Activated by Proteolysis
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R15-I16
Chymotrypsinogen (inactive)
-Chymotrypsin (active)
S14-R15 T147-N148
Trypsin
-Chymotrypsinogen (active)
-Chymotrypsin
I16L13 A149Y146
Disulfide bonds
Ch
arg
e R
ela
y in
Active S
ite
Ser195
His 57
Asp 102
H–O–CH2
O
C–O -
=
Active Ser
H–N N
C C
C
H
H
CH2
Ser195
His 57
Asp 102
- O–CH2
OC–O–H
=
N N–H
C C
C
H
H
CH2
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pH Influences Chymotrypsin Activity
5 6 7 8 9 10 11
pH
Relative
Activity
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162
pH Influences N
et Charge of P
rotein Jua
ng
RH
(2
00
4)
BC
ba
sics
+Net Charge of a Protein
Buffer pH
Isoelectric point,pI
-
3
4
5
6
7
8
9
10
0+
Imidazole on Histidine Is Affected by pH
H–N N
C C
C
H
H
H+
pH 6 pH 7
+H–N N–H
C C
C
H
H
Inactive+ Ser
195
His 57
Asp 102
H–O–CH2
O
C–O -
=
H–N N–H
C C-H
C
CH2
H
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158
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Chymotrypsin Produces New Ile16 N-Terminal
I16L13 Y146
Asp 194
–CH2COO-
Ile 16NH2–
Ile 16+ NH3–
5 6 7 8 9 10 11pH
Relative activity
pH 9 pH 10pKa
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165
New NH2-terminus
New Ile16 N-Terminal Stabilizes Asp194
Asp 102
His 57 Ser 195
Asp 194
Gly 193
Ile 16
+NH3
Catalytic Triad
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.206
Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.112
O (CH3)2CH–O– P –O–CH(CH3)2
F
=
Chymotrypsin Ser195 Inhibited by DIFP
Diisopropyl-fluorophosphate (DIFP)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
O-…H
CH2
Ser 195
O (CH3)2CH–O– P –O–CH(CH3)2
=
O
CH2
Ser 195
XXXX
Addition of Substrate Blocks DIFP Inhibition
Reaction time
Percent Inhibition of activity (%
)
100
50
0
No substrate
Add substrate
S
+ DIFP
+ DIFP & substrate
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
XXXX
Chymotrypsin Also Catalyzes Acetate
O-C N- H
O-C O-
Peptide bond
Ester bond
O
CH3–C–O– –NO2
Nitrophenol acetate
HO– –NO2
O
CH3–C–OH
Hartley & Kilby
Chymotrypsin+ H2O
Nitrophenol
Acetate
No acetate was detected at early stage
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168
O -
C
Time (sec)N
itrop
hen
ol
Two-Stage Catalysis of Chymotrypsin
O
CH3–C–O– –NO2
Nitrophenol acetate
OC
O
CH3–C HO– –NO2
+ H2O
O-HC
CH3COOH
Kinetics of reaction
Two-phasereaction
Acylation
Deacylation (slow step)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169
Extra Negative Charge Was Neutralized
O-C N- H
O-C-OH
NH2-
-C-C-N-C-C-N-C-C-N- H H
E + S
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179
O -
-C N- HO H
O -
-C N- HO H