Characterization of ScPrI, a small serine protease, from mycelia of Schizophyllum commune

Schitophyllum commune produces a variety of mycelial proteolytic enzymes. T he specific functions of many of these enzymes are unknown, but several hav e elevated activity when the mycelium is grown in nitrogen-limiting conditi ons, suggesting a role in mycelial autolysis. We have purified one of these nitrogen-limitation induced enzymes, a small serine protease, ScPrI, from S. commune mycelial extracts. ScPrI has an apparent molecular mass of 22 kD a and is active against classical substrates for chymotrypsin and subtilisi n proteases. The pH optimum for activity is neutral to slightly alkaline an d the protein denatures above 50 degrees C. The enzyme is inhibited by PMSF , TPCK and chymostatin, and it shows little dependence on metal ions. Hydro lysis of oxidized insulin B-chain peptide by ScPrI demonstrated cleavage fo llowing aromatic amino acids and leucine. Kinetic analysis of hydrolysis of N-succinyl-AAPF-pNA and N-succinyl-AAPL-pNA revealed similar K(m)s for bot h substrates but the V-max was nearly 3-fold higher for the substrate with phenylalanine in the P1 position.