DELOCALIZING TRYPSIN SPECIFICITY WITH METAL ACTIVATION

Recognition for proteolysis by trypsin depends almost exclusively on t ight binding of arginine or lysine side chains by the primary substrat e specificity pocket. Although extended subsite interactions are impor tant for catalysis, the majority of binding energy is localized in the P-1 pocket. Analysis of the interactions of trypsin with the P-1 resi due of the bound inhibitors ecotin and bovine pancreatic trypsin inhib itor suggested that the mutation D189S would improve metal-assisted tr ypsin N143H, E151H specificity toward peptides that have a Tyr at P-1 and a His at P-2'. In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved towar d the tyrosine-containing peptide AGPYAHSS. Trypsin N143H, E151H, D189 S exhibits a 25-fold increase in activity with nickel and a 150-fold i ncrease in activity with zinc relative to trypsin N143H, E151H on this peptide. In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, n ickel, and zinc. With this substrate, copper yields a 30-fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hy drolysis without metal. These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delo calize protease specificity by increasing relative substrate binding c ontributions from alternate engineered subsites.