Relocating a negative charge in the binding pocket of trypsin.

The functional and structural consequences of altering the position of the negatively charged aspartate residue at the base of the specificity pocket of trypsin have been examined by site-directed mutagenesis, kinetic characterization and crystallographic analysis. Anionic rat trypsin D189G/G226D exhibits a high level of catalytic activity on activated amide substrates, but its relative preference for lysine versus arginine as the P1 site residue is shifted by 30 to 40-fold in favor of lysine. The crystal structure of this variant has been determined in complexes with BPTI (bovine pancreatic trypsin inhibitor), APPI (amyloid beta-protein precursor inhibitor domain) and benzamidine inhibitors, at resolutions of 2.1 A, 2.5 A and 2.2 A, respectively. Asp226 bridges the base of the specificity pocket with its negative charge partially buried by interactions made with Ser190 and Tyr228. An equal reduction in the affinity of the variant enzyme for Arg and Lys substrates is attributable to a decreased electrostatic interaction of each ligand with the relocated aspartate residue. Comparison of structural and functional parameters with those of wild-type trypsin suggests that direct hydrogen-bonding electrostatic contacts in the S1 site do not significantly improve the free energy of substrate binding relative to indirect water-mediated interactions. The conformation adopted by Asp226, as well as by other adjacent side-chain and backbone groups, depends upon the ligand bound in the primary specificity pocket. This structural flexibility may be of critical importance to the retention of catalytic activity by the variant enzyme.