Two approaches are proposed to investigate substrate specificity and catalytic mechanisms of two different enzymes, citrate synthase (CS) and serine carboxypeptidase (CPD). The first will be an X-ray crystallographic analysis of the structures of the enzymes and to compare ligand affinities and catalytic activity of related or mutant forms of the enzymes. The second will be to investigate the role of electrostatic fields in the mechanisms by use of theoretical calculations. Citrate synthase is one of few enzymes capable of directly forming a carbon bond. A detailed mechanism for part of the reaction has been proposed. Site-directed mutants of CS with differing activity have been constructed. Their structures will be analyzed by crystallographic techniques, enzyme kinetics and electrostatic field calculations to study the roles of selected side chains. Serine carboxypeptidases are highly specific proteinases which are essential for the maturation of peptide hormones. They are unrelated to other serine proteinases, but have the same active site groups. Analysis of inhibitor binding will determine the basis for substrate specificity. Selected mutants of yeast CPD will be studied to determine the basis for the unusual pH dependence of the reaction and to test whether the enzymatic mechanism is similar to that proposed for other serine proteinases.
