Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Enzymatic Hydrolysis and Group Transfer Reactions 85
Figure 5.4 Active site of bovine trypsin (PDB Wle 1BTX), showing in red (from left to right) the
catalytic triad Asp-102, His-57 and Ser-195; the oxyanion hole formed by Gly-193; and a bound
boronic acid inhibitor in black.
hydrogen bonds between the tetrahedral oxyanion and the backbone amide
N2H bonds of Ser-195 and Gly-193, as shown in Figure 5.5. Note that these
interactions are speciWc for the oxyanion intermediate and are not formed with
the bound substrate, leading to reduction of the activation energy for the
overall step (since this is a non-isolable high-energy intermediate that is similar
to the transition state for the Wrst step, the transition state stabilisation logic of
Section 3.4 applies in this case).
Protonation of the departing nitrogen by His-57 gives the amine product
and leads to the formation of an acyl enzyme intermediate. There is considerable
experimental evidence for the existence of this covalent intermediate from
kinetic studies, trapping experiments and X-ray crystallography. Histidine-57
then acts as a base to deprotonate water attacking the acyl enzyme intermediate,
leading to a second tetrahedral oxyanion intermediate which is once again
stabilised by hydrogen bond formation. Protonation of the departing serine
oxygen by His-57 leads to release of the carboxylic acid product and completion
of the catalytic cycle.
The use of the hydroxyl group as a nucleophile in this reaction is quite
remarkable since an alcohol would normally have a pK a of about 16, whereas
the pK a of histidine is normally in the range 6–8. EVectively this enzyme is using