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10388 Biochemistry 1991, 30, 10388-10398 Structural Basis for Broad Specificity in a-Lytic Protease Mutants+,$

Summary: 10388 Biochemistry 1991, 30, 10388-10398
Structural Basis for Broad Specificity in a-Lytic Protease Mutants+,$
Roger Bone,sJ Amy Fujishige,i Charles A. Kettner,I and David A. Agard*,(
Departments of Biochemistry and Biophysics and of Pharmaceutical Chemistry and The Howard Hughes Medical Institute,
University of California at San Francisco, San Francisco, California 94143-0448, and Du Pont Merck Pharmaceutical
Company, Du Pont Experimental Station, Wilmington, Delaware 19880-0328
Received February 25, 1991; Revised Manuscript Received June 25, 1991
ABSTRACT: Binding pocket mutants of a-lytic protease (Met 192 -Ala and Met 213 -Ala) have been
constructed recently in an effort to create a protease specific for Met just prior to the scissile bond. Instead,
mutation resulted in proteases with extraordinarily broad specificity profiles and high activity [Bone, R.,
Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-1951. To understand the structural basis for the
unexpected specificity profiles of these mutants, high-resolution X-ray crystal structures have been determined
for complexes of each mutant with a series of systematically varying peptidylboronic acids. These inhibitory
analogues of high-energy reaction intermediates provide models for how substrates with different side chains
interact with the enzyme during the transition state. Fifteen structures have been analyzed qualitatively
and quantitatively with respect to enzyme-inhibitor hydrogen-bond lengths, buried hydrophobic surface
area, unfilled cavity volume, and the magnitude of inhibitor accommodating conformational adjustments
(particularly in the region of another binding pocket residue, Val 217A). Comparison of these four parameters
with the Kiof each inhibitor and the k,, and K,,,of the analogous substrates indicates that while no single


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


Collections: Biotechnology; Biology and Medicine