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 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.
D001896:Boron Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005998:Glycine; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001896:Boron Compounds; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase; D005998:Glycine