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Enhancing the catalytic activity of an engineered threonine protease

In this study, the Cys‐42—Cys‐58 disulfide bridge of S195T trypsin was replaced with two valine residues to: 1) increase threonine protease activity and 2) better understand the structual limitations and requirements of this subsite with respect to catalysis. The activities of C42V/C58V and C42V/C58V/S195T trypsins (VV‐Tn and VVT‐Tn, respectively) were evaluated with benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) p‐nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) substrates. VVT‐Tn was 150 and 10 times more active towards the pNA and AMC substrates, respectively, than C42A/C58V/S195T trypsin (AVT‐Tn; kcat/KM values). In a thermostability study, both variants and WT trypsin retained > 80% of their activity suggesting that the substitutions did not destabilize the enzymes. The pH dependence of activity profiles were identical to WT trypsin, suggesting that any structural differences do not perturb the pKas of catalytically relevant residues. Finally, the KI of p‐aminobenzamidine was identical for native and VV‐Tn but that of VVT‐Tn was half the KI for WT. Therefore, Thr‐195 may affect the primary binding pocket structure. Collectively, these results indicate that the activity of Thr‐195 trypsin variants may be increased by appropriate substitutions at positions 42 and 58. However, the structural pertubations induced by these substitutions make achievement of wild‐type levels of activity less likely.