IDENTIFICATION OF ACTIVE-SITE RESIDUES OF THE TSP PROTEASE

In a search for active-site residues of the Tsp protease, 20 positions were individually mutated to alanine, the mutant strains were assayed for growth defects in vivo, and the purified proteins were assayed fo r proteolytic activity in vitro. Alanine substitutions at three positi ons, Ser-430, Asp-441, and Lys-455, result in inactive proteases that have structures and substrate-binding properties similar to wild type, suggesting that the side chains at these positions participate in cat alysis. Replacing Ser-430 with cysteine results in a partially active protease, which is inhibited by cysteine-modifying reagents. Replacing Asp-44l with asparagine does not significantly affect activity. Howev er, other residues, including histidine and arginine, cannot functiona lly replace Lys-455. These data are consistent with a serine-lysine dy ad mechanism, similar to those proposed for the LexA-like proteases, t he type I signal peptidases, and the class A beta-lactamases.