Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Methods for Studying Enzymatic Reactions 75
< 1:1. For a discussion of heavy atom isotope eVects the reader is referred to
specialist references.
4.7 Determination of active site catalytic groups
As well as examining the molecular details of an enzymatic reaction, it is
equally important to study the groups present in the enzyme active site which
carry out the catalysis.
One convenient method for obtaining clues regarding active site catalytic
groups is to analyse the variation of enzyme activity with pH: a pH/rate proWle.
Thus, if there are acidic and basic groups involved in the enzyme mechanism,
they must be in the correct ionisation state in order for the enzyme to operate
eYciently. For example, the ketosteroid isomerase reaction illustrated above
(Figure 4.27) has the pH/rate proWle shown in Figure 4.28, from which the pK a
value of 4.7 for the active site Asp-38 was Wrst deduced.
The second method that can be used to identify active site groups is by
covalent modiWcation. There are a series of chemical reagents available which
will react in a fairly speciWc way with diVerent amino acid side chains, shown in
Table 4.3. Thus, if an enzyme is inactivated upon treatment with diethyl
pyrocarbonate, then this provides a clue that there may be an essential histidine
residue at the active site of the enzyme. However, residues identiWed by such
methods are not necessarily catalytic groups: they may simply be residues in the
vicinity of the active site which when covalently modiWed block the entrance to
the active site suYciently to inactivate the enzyme.
A related technique involving substrate analogues is known as aYnity
labelling. A substrate analogue is synthesised containing a reactive functional
group (e.g. halogen substituent, epoxide, etc.) in a part of the molecule.
The substrate analogue is recognised by the enzyme and binds to the active
site in the same way as the natural substrate, but then alkylates an essential
enzyme
activity
4.7
pH
Figure 4.28 pH/rate proWle of ketosteroid isomerase.