Kinetic analysis of enzyme mutants for identification of catalytic residues

Abstract

Abstract This article will provide a guide for the experimentalist who wishes to define the specific roles of active site residues in catalysis by the enzyme that he or she is studying. This guide is based on the authors’ own experience in analysing the catalytic residues of Pseudomonas sp. strain CBS-3 4-chlorobenzoyl-CoA dehalogenase using a site-directed mutagenesis-transient kinetic analysis based approach. General methods that one might apply to any enzyme will be illustrated with experiments carried out with the dehalogenase.