Abstract：
:The specificity of trypsin for Arg- and Lys-containing substrates depends upon the presence of Asp189 at the base of the primary binding pocket. The crystal structure of anionic rat trypsin D189S complexed with BPTI reveals that removal of the aspartate side chain permits the binding of a well-ordered acetate ion in a similar position. The acetate makes polar interactions with Gly226, Tyr228, and several water molecules and is further accommodated by rotation of the Ser189 side chain out of the binding pocket. The carboxylate group of the acetate anion is oriented toward the substrate in a manner similar to that of Asp189 and Asp226 in wild-type trypsin and trypsin D189G/G226D. Evaluation of kinetic parameters for amide substrate cleavage by trypsin D189S shows that high concentrations of acetate increase the catalytic efficiency of the enzyme by as much as 300-fold. Under these conditions, the rate of substrate turnover toward a peptidylarginine amide substrate equals that of wild-type trypsin. These data demonstrate that the well-established requirement for a negatively charged moiety at the base of the trypsin specificity pocket may be fulfilled by a noncovalently bound ligand. The binding pocket of this variant maintains a trypsin-like conformation, explaining the inability of the mutant enzyme to efficiently hydrolyze chymotrypsin substrates possessing Phe in the P1 position.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Perona JJ,Hedstrom L,Wagner RL,Rutter WJ,Craik CS,Fletterick RJdoi
10.1021/bi00177a016subject
Has Abstractpub_date
1994-03-22 00:00:00pages
3252-9issue
11eissn
0006-2960issn
1520-4995journal_volume
33pub_type
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