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86 Chapter 5
His 57
His 57
Asp 102
O −
O
H
N
N
R'
Ser 195
H O
O
NH
RNH Ph
− +
Asp 102 O H N N
Ser 195
H O
H
O-
O
NH
R'
H
RNH Ph
Ser 195
N
Gly 193
N
R'NH 2
His 57
His 57
O − +
Asp 102 H N N
Ser 195
H O
H
O−
O
HO
H
RNH Ph
Ser 195
N
Gly 193
N
Asp 102
O −
O
H
N
N
H
O
H
Ser 195
O
RNH
O
Ph
His 57
Asp 102
O −
O
H
N
N
H
HO
O
Ser 195
O
RNH
Ph
Figure 5.5 Mechanism for a-chymotrypsin.
a relatively weak imidazole base to generate a potent alkoxide nucleophile. This
process is made thermodynamically favourable by participation of Asp-102 in
stabilising the imidazolium cation of His-57. Mutant enzymes have been generated
using site-directed mutagenesis in which Asp-102 has been replaced by
asparagine – these enzymes are 10 4 -fold less active, showing the catalytic
importance of this interaction.
Serine proteases are speciWcally inactivated by two classes of inhibitors:
organophosphorus and chloromethyl ketone substrate analogues. Mechanisms
of inactivation are shown in Figure 5.6. Organophosphorus inhibitors are
attacked by the active site serine, generating stable tetrahedral phosphate esters
which closely resemble the tetrahedral transition state of the normal enzymatic
reaction, and hence are bound tightly by the enzyme. Chloromethyl ketone
analogues were found to modify the active site histidine base, in this case