Kinetic and structural characterization of mutations of glycine 216 in alpha-lytic protease: a new target for engineering substrate specificity

J Mol Biol. 1995 Dec 8;254(4):720-36. doi: 10.1006/jmbi.1995.0650.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Boron Compounds / chemistry
  • Boron Compounds / metabolism
  • Boron Compounds / pharmacology
  • Crystallography, X-Ray*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Glycine*
  • Kinetics
  • Macromolecular Substances
  • Models, Molecular
  • Mutagenesis
  • Mutation*
  • Protein Conformation
  • Serine Endopeptidases / chemistry*
  • Serine Endopeptidases / genetics*
  • Serine Endopeptidases / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • Boron Compounds
  • Enzyme Inhibitors
  • Macromolecular Substances
  • Serine Endopeptidases
  • myxobacter alpha-lytic proteinase
  • Glycine