Mapping the active site of papain with the aid of peptide substrates and inhibitors

Abstract The active site of an enzyme performs the twofold function of binding a substrate and catalysing a reaction. The efficiency of these actions determines the overall activity of the enzyme towards the particular substrate, i.e. determines the specificity of the enzyme. It is therefore possible to obtain information on the active site by the kinetics of the enzyme’s reactions with different substrates and inhibitors. An important feature of the active site is its size. It should be possible to 'measure’ this by using substrates or inhibitors large enough to show up the interactions of the furthermost parts of the binding site. In the present series of investigations on proteolytic enzymes, our approach is to compare the activity of the enzyme towards (a) peptides of increasing length, (b) diastereoisomeric pairs of peptides in which a particular amino acid residue has been replaced by its antipode, and (c) pairs of substrates in which a particular side chain (say a methyl group) has been replaced by another (say an aromatic group). The influence of these changes on reaction rates as a function of distance from the point of cleavage indicates the extent of the active site (Schechter, Abramowitz & Berger 1965; Abramowitz, Schechter & Berger 1967).