Format

Send to

Choose Destination
J Mol Biol. 1995 Dec 8;254(4):720-36.

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

Author information

1
Howard Hughes Medical Institute, University of California, San Francisco 94143-0448, USA.

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.

PMID:
7500345
DOI:
10.1006/jmbi.1995.0650
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center