The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2.

Variation in the kinetic parameters, kcat and Km, with pH has been used to obtain evidence for significant acid-dissociation processes in the hydrolysis of octapeptide substrates by three aspartic proteinases. These substrates are all cleaved at the peptide bond between a Phe (P1) and a p-nitroPhe (P1') residue resulting in a shift in absorbance at 300 nm that facilitates kinetic measurements. The substrates differ in the amino-acid residues present in the P3 and the P2 positions. Porcine pepsin, calf chymosin, and the aspartic proteinase from Endothia parasitica all show pH dependencies that imply that favorable or unfavorable interactions can occur with the S3 or S2 areas of the enzyme-active site. Examination of the crystallographically determined structure of the E. parasitica proteinase and consideration of the amino-acid sequence differences between the three enzymes suggests that the origin of the pH effects arises from favorable interactions between Glu-13 (COO-) of pig pepsin and Thr (OH) or His (ImH+) in P3 of a substrate. Similarly, Lys-220 (NH3+) of chymosin and a Glu (COO-) in P2 of a substrate may produce a favorable interaction and Asp-77 (COO-) of E. parasitica proteinase and a Glu (COO-) in P2 of a substrate may produce an unfavorable interaction. These results lead to possible explanations for subtle specificity differences within a family of homologous enzymes, and suggest loci for study by site-directed mutagenesis.