The specificity of two proteinases that cleave adjacent to arginine, Cl esterase and acrosin, for peptide p-nitroanilide substrates

Abstract Relative values of V max / K m for hydrolysis of 40 peptide p -nitroanilides catalyzed by human Cl s ¯ and human acrosin are reported. For Cl s ¯ , Ac-Lys(γCbz)-Gly-Arg is the optimum sequence, but 25% of the substrateshave ( V max / K m ) rel 0.25 compared to this sequence. The best acrosin substrate tested has the sequence Tos-Gly-Pro-Arg, although ( V max / K m ) rel 0.15 for more than half of the substrates. Proline at P 2 is preferred by acrosin. Both enzymes prefer arginine at P 1 ⩾ 3-fold over lysine and will not accept citrulline. In addition, occupancy of site S 3 may yield an increase in V max / K m of ⩾ 10-fold with either enzyme, but many residues are accepted at S 2 , S 3 and S 4 . Thus, an acrosin assay using Tos-Gly-Pro-Arg p -nitroanilide as a substrate is more than 20-times as sensitive as existing assays with blocked arginine derivatives.