The catalytic triad of rat anionic trypsin has been systematically altered by site-directed mutagenesis to determine the activity of alternate combinations of aminoacids toward the hydrolysis of peptide bonds. Genetically modified rat trypsins H57A, H57D, H57E, H57K, H57R, H57A/D102N, H57D/D102N, H57L/D102N, H57K/D102N, D102N, S195A, S195T, and H57A/D102N/S195A have been generated. Rigorous steps were taken to show that the resultant catalysis was due to the mutant enzymes and not contaminants. Each of the variants exhibit measurable activity toward the activated amide substrate Z-GPR-AMC. At pH 8.0 kal ranges from 0.011 to 1.3 min" 1 (0.0004-0.04% of wild-type). At pH 10.5/ccat ranges from 0.012 to 140 m" 1 (0.0004-5% of wild-type). Themutant trypsins were subsequently assayed for their ability to hydrolyze the unactivated amide linkages of protein substrates. Trypsins D102N, H57K, and H57K/D102N exhibited the highest level of activity. The kat for the D102N enzyme was 4 h'1 (0.003% of wild-type). The H57A/D102N double mutant was not as active but was chosen for further study since it was the simplest trypsin to exhibit peptidase activity. Its kM was~ 0.1-0.2 h'1 at pH 8.0 and 0.7 h" 1 at pH 10.1. These experiments demonstrate that an intact catalytic triad is not a requirement for peptide bond cleavage and that designed serine peptidases need not include a catalytic histidine or aspartic acid.

The development of peptidases with designed specificities would facilitate the manipulation of peptides and proteins. The challenge in designing such catalysts is the inclusion of interdependent binding and catalytic motifs within a common structural framework to achieve the energetically demanding hydrolysis of peptide bonds. 1 Initial studies have involved the derivatization of small molecules with reactive moieties to partially or fully mimic the chemistry of the serine protease catalytic triad. These catalysts have helped elucidate some aspects of the interactions between members of the triad2· 3 but have not yet been shown to catalyze the cleavage of amide linkages. Recently, this approach has been extended with a de novo designed four helix bundle polypeptide