In a previous successful attempt to convert trypsin to a chymotrypsin-like protease, 15 residues of trypsin were replaced with the corresponding ones in chymotrypsin. This suggests a complex mechanism of substrate recognition instead of a relatively simple one that only involves three sites, residues 189, 216 and 226. However, both trypsin®elastase and chymotrypsin®trypsin conversion experiments carried out according to the complex model resulted in non-speci®c proteases with low catalytic

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... ity. Chymotrypsin used in the latter studies was of B-type, containing an Ala residue at position 226. Trypsins, however, contain a conserved Gly at this site. The substantially decreased trypsin-like activity of the G226A trypsin mutant also suggests a speci®c role for this site in substrate binding. Here we investigate the role of site 226 by introducing the A226G substitution into chymotrypsin®trypsin mutants which were constructed according to both the simple (S189D mutant) and the complex model (S 1 mutant) of speci®city determination. The kinetic parameters show that the A226G substitution in the S 1 mutant increased the chymotrypsin-like activity, while the trypsin-like activity did not change. In contrast, this substitution in the S189D chymotrypsin mutant resulted in a 100-fold increase in trypsin-like activity and a trypsin-like speci®city pro®le as tested on a competing oligopeptide substrate library. Additionally, the S189D+A226G mutant is the ®rst trypsin-like chymotrypsin that undergoes autoactivation, an exclusive property of trypsinogen among pancreatic serine proteases.