Studies of binding sites in the subtilisin from Bacillus lentus by means of site directed mutagenesis and kinetic investigations.

Abstract

The binding site of a proteolytic enzyme may be divided into a number of subsites, each by multiple interactions securing the binding of a single amino acid residue and the proper alignment of the substrate prior to catalysis. The properties of the amino acid residues which constitute a given binding subsite determine which amino acid residue(s) of the substrate may bind and thus, they provide the basis of subsite specificity. The nature of these interactions may be studied by various techniques but it is a prerequisite that the individual subsites are carefully mapped by kinetic investigations using systematic variations of substrate structures. The recent development of highly efficient donor/acceptor pairs for substrates based on intramolecular fluorescence quenching, allowing the use of long peptide substrates spanning the entire binding site, represents a significant improvement in this context.