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JMB--MS 667 Cust. Ref. No. FC029/95 [SGML] J. Mol. Biol. (1995) 251, 116134

Summary: JMB--MS 667 Cust. Ref. No. FC029/95 [SGML]
J. Mol. Biol. (1995) 251, 116134
Functional Linkage Between the Active Site of a-Lytic
Protease and Distant Regions of Structure: Scanning
Alanine Mutagenesis of a Surface Loop Affects
Activity and Substrate Specificity
James E. Mace*, Barry J. Wilk and David A. Agard
Previous structural and kinetic characterization of mutations within theHoward Hughes Medical
active site of a-lytic protease have demonstrated that amino acid residues inInstitute and Department of
direct contact with the substrate are major substrate specificity determinants.Biochemistry and Biophysics
The experiments described here identify residues 216226 of a-lytic proteaseUniversity of California
San Franciso, CA as a region of structure peripheral to the active site that also plays an
important role in establishing the substrate specificity of the enzyme.94143-0448, USA
Alanine substitution mutations within this surface loop of 19 amino acid
residues significantly perturb the enzyme's specificity profile, despite being
as far as 21 from the hydroxyl group of Ser195. The kinetic consequences
of the mutations are remarkably independent of position within the loop
and suggest that active site plasticity is affected more than static struc-
ture. Kinetic characterization of double mutants with the Met190:Ala
broad-specificity active site mutation reveals varying degrees of non-


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


Collections: Biotechnology; Biology and Medicine