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J. Mol. Biol. (1995) 254, 720736 Kinetic and Structural Characterization of Mutations
 

Summary: J. Mol. Biol. (1995) 254, 720736
Kinetic and Structural Characterization of Mutations
of Glycine 216 in a-Lytic Protease: A New Target for
Engineering Substrate Specificity
James E. Mace and David A. Agard*
Gly216 in the active site of the broadly specific MA190 mutant of a-lyticHoward Hughes Medical
Institute and Department of protease has been found to be remarkably tolerant of amino acid
Biochemistry and Biophysics substitutions. Side-chains as large as Trp can be accommodated within the
substrate-binding pocket without abolishing catalysis, and have majorUniversity of California
San Francisco effects upon the substrate specificty of the enzyme. Kinetic characterization
of eleven enzymatically active mutants against a panel of eight substratesCA 94143-0448, USA
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-para-
metric statistical analysis of structural variation among these enzyme:
inhibitor complexes is presented. The roles of active site plasticity and
dynamics in a-lytic protease function and substrate preference are also
addressed. The results strongly suggest that substrate specificity

  

Source: Agard, David - Department of Biochemistry and Biophysics, University of California at San Francisco

 

Collections: Biotechnology; Biology and Medicine