KINETIC AND STRUCTURAL CHARACTERIZATION OF MUTATIONS OF GLYCINE-216 IN ALPHA-LYTIC PROTEASE - A NEW TARGET FOR ENGINEERING SUBSTRATE-SPECIFICITY

Gly216 in the active site of the broadly specific MA190 mutant of alph a-lytic protease has been found to be remarkably tolerant of amino aci d substitutions. Side-chains as large as Trp can be accommodated withi n the substrate-binding pocket without abolishing catalysis, and have major effects upon the substrate specificity of the enzyme. Kinetic ch aracterization of eleven enzymatically active mutants against a panel of eight substrates clearly revealed the functional consequences of th e substitutions at position 216. To understand better the structural b asis for their altered specificity, the GA216 + MA190 and GL216 + MA19 0 mutants have been crystallized both with and without a representativ e series of peptide boronic acid transition-state analog inhibitors. A n empirical description and non-parametric statistical analysis of str uctural variation among these enzyme:inhibitor complexes is presented. The roles of active site plasticity and dynamics in alpha-lytic prote ase function and substrate preference are also addressed. The results strongly suggest that substrate specificity determination in alpha-lyt ic protease is a distributed property of the active site and substrate molecule. (C) 1995 Academic Press Limited