An Investigation into the Minimum Requirements for Peptide Hydrolysis by Mutation of the Catalytic Triad of Trypsin

The catalytic triad of rat anionic trypsin has been systematically altered by site-directed mutagenesis to determine the activity of alternate combinations of amino acids 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 k_cat ranges from 0.011 to 1.3 min^-1 (0.0004–0.04% of wild-type). At pH 10.5 k_cat ranges from 0.012 to 140 m^-1 (0.0004–5% of wild-type). The mutant 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 k_cat 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.