the mechanism underlying the binding of a substrate(s) to the enzyme catalytic site
and the subsequent conversion of the substrate(s) to product(s). The mechanism must
include details of the nature of the binding and catalytic sites, the nature of the
intermediate enzyme–substrate complex(es), and the associated electronic and stereo-
chemical events that result in the formation of the product. A wide range of strategies
and analytical techniques has been adopted to gain such an understanding:- X-ray crystallographic studies:These are capable of giving, either directly or indirectly,
decisive information about the mechanism of enzyme action. X-ray diffraction patterns
enable the position of each amino acid in the protein to be located and the details deduced
of how the substrate binds and undergoes reaction. Such deductions are facilitated by the
study of crystals grownin the presenceandabsence of thesubstrate,competitive inhibitor
or of effector molecules. The Catalytic Site Atlas (www.ebl.ac.uk/thornton-srv/databases)
and Protein Data Bank (www.wwpdp.org) list the active sites and catalytic residues of
enzymes whose three-dimensional structure has been determined. As knowledge of
protein structures and catalytic mechanisms has increased, computer programs have
become available that enable the chemical and stereochemical conformations of the
substrate(s) to be modelled and a prediction made of the three-dimensional structure
of the enzyme that promotes the formation of product(s). This approach is now widely
used in the pharmaceutical industry to identify ‘lead’ compounds for the development
of new drugs (Section 18.2). - Irreversible inhibitor and affinity label studies:Irreversible inhibitors act by forming
a covalent bond with the enzyme. By locating the site of the binding of the inhibitor,
information can often be obtained about the identity of specific amino acids in the
binding site. A development of this approach is the use ofphotoaffinity labelsthat
structurally resemble the substrate but which contain a functional group, such as azo
(N¼N), which on exposure to light is converted to a reactive functional group, such
as a carbene or nitrene, which forms a covalent bond with a neighbouring functional
group in the active site. It is common practice to tag the inhibitor or photoaffinity label
with a radioisotope so that its location in the enzyme protein can easily be established
experimentally. - Kinetic studies:This approach is based on the use of a range of substrates and/or
competitive inhibitors and the determination of the associatedKm,kcatandKivalues.
These allow correlations to be drawn between molecular structure and kinetic
constants and hence deductions to be made about the structure of the active site.
Further information about the structure of the active site can be gained by studying
the influence of pH on the kinetic constants. Specifically, the effect of pH onKm
(i.e. on binding of E to S) and onVmaxorkcat(i.e. conversion of ES to products)
is studied. Plots are then made of the variation of logKmwith pH and of logVmax
or logkcatwith pH. The intersection of tangents drawn to the curves gives an
indication of the pKavalues of ionisable groups involved in the active site (Fig 15.8).
These are then compared with the pKavalues of the ionisable groups known to
be in proteins. For example, pH sensitivity around the range 6–8 could reflect
the importance of one or more imidazole side chains of a histidine residue in the
active site.
612 Enzymes