Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A. Dependence on pH and effect of enzyme phosphorylation level.

Steady-state kinetic parameters for the hydrolysis of the chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine gastricsin and pepsin A were determined. It was shown that the phosphate content of bovine pepsin A is without any significant effect on that parameters. At pH 4.7, the specificity constant (kcat/Km) was 2455 and 2150 mM-1 X s-1 for the most phosphorylated bovine pepsin A (2.58 phosphate groups per molecule), before and after treatment by potato acid phosphatase, respectively. The kcat/Km ratio found for bovine gastricsin (1314 mM-1 X s-1) was closer to that of bovine pepsin A than that previously reported for chymosin (25 mM-1 X s-1). The spectral properties of the chromophoric tripeptide Leu-Ser-Phe(NO2) in the pH range 1-3.6 were investigated. We have shown that the hexapeptide hydrolysis could be followed by difference spectrophotometry at 295 nm (delta epsilon = -235 M-1 X cm-1 at pH 1.0) thus allowing to study the effect of pH on bovine pepsin A activity in a pH range which could not be explored earlier. The pH-dependence of kcat/Km ratio of unphosphorylated bovine pepsin A indicated that enzyme activity was dependent upon the ionization of two groups of the enzyme whose pK are 1.2 and 5.0. These pK values strongly suggest the involvement of two carboxyl groups probably corresponding to the two reactive aspartyl residues (Asp32 and Asp215) identified through active site-directed reagents for all the aspartic proteinases so far tested.