Dissecting the catalytic triad of a serine protease

Abstract

Serine proteases are present in virtually all organisms and function both inside and outside the cell¹; they exist as two families, the 'trypsin-like' and the 'subtilisin-like', that have independently evolved a similar catalytic device² characterized by the Ser, His, Asp triad, an oxyanion binding site, and possibly other determinants that stabilize the transition state (Fig. l) ^2–4. For Bacillus amyloliquefaciens subtilisin, these functional elements impart a total rate enhancement of at least 10⁹ to 10¹⁰ times the non-enzymatic hydrolysis of amide bonds. We have examined the catalytic importance and interplay between residues within the catalytic triad by individual or multiple replacement with alanine(s), using site-directed mutagenesis^5,6 of the cloned B. amyloliquefaciens subtilisin gene⁷. Alanine substitutions were chosen to minimize unfavourable steric contacts and to avoid imposing new charge interactions or hydrogen bonds from the substituted side chains. In contrast to the effect of mutations in residues involved in substrate binding^8–10, the mutations in the catalytic triad greatly reduce the turnover number and cause only minor effects on the Michaelis constant. Kinetic analyses of the multiple mutants demonstrate that the residues within the triad interact synergistically to accelerate amide bond hydrolysis by a factor of ∼ 2×l0⁶.