EXOGENOUS ACETATE RECONSTITUTES THE ENZYMATIC-ACTIVITY OF TRYPSIN ASP189SER

The specificity of trypsin for Arg- and Lys-containing substrates depe nds upon the presence of Asp189 at the base of the primary binding poc ket. The crystal structure of anionic rat trypsin D189S complexed with BPTI reveals that removal of the aspartate side chain permits the bin ding of a well-ordered acetate ion in a similar position. The acetate makes polar interactions with Gly226, Tyr228, and several water molecu les and is further accommodated by rotation of the Ser189 side chain o ut of the binding pocket. The carboxylate group of the acetate anion i s 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 show s that high concentrations of acetate increase the catalytic efficienc y of the enzyme by as much as 300-fold. Under these conditions, the ra te of substrate turnover toward a peptidylarginine amide substrate equ als that of wild-type trypsin. These data demonstrate that the well-es tablished requirement for a negatively charged moiety at the base of t he trypsin specificity pocket may be fulfilled by a noncovalently boun d ligand. The binding pocket of this variant maintains a trypsin-like conformation, explaining the inability of the mutant enzyme to efficie ntly hydrolyze chymotrypsin substrates possessing Phe in the P1 positi on.