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, 254 (4), 720-36

Kinetic and Structural Characterization of Mutations of Glycine 216 in Alpha-Lytic Protease: A New Target for Engineering Substrate Specificity

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Kinetic and Structural Characterization of Mutations of Glycine 216 in Alpha-Lytic Protease: A New Target for Engineering Substrate Specificity

J E Mace et al. J Mol Biol.

Abstract

Gly216 in the active site of the broadly specific MA190 mutant of alpha-lytic protease has been found to be remarkably tolerant of amino acid substitutions. Side-chains as large as Trp can be accommodated within the substrate-binding pocket without abolishing catalysis, and have major effects upon the substrate specificity of the enzyme. Kinetic characterization of eleven enzymatically active mutants against a panel of eight substrates clearly revealed the functional consequences of the substitutions at position 216. To understand better the structural basis for their altered specificity, the GA216 + MA190 and GL216 + MA190 mutants have been crystallized both with and without a representative series of peptide boronic acid transition-state analog inhibitors. An empirical description and non-parametric statistical analysis of structural variation among these enzyme: inhibitor complexes is presented. The roles of active site plasticity and dynamics in alpha-lytic protease function and substrate preference are also addressed. The results strongly suggest that substrate specificity determination in alpha-lytic protease is a distributed property of the active site and substrate molecule.

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