Abstract
The kinetics of S-PI (Pepstatin Ac)-insensitive acid proteinases A-2 and C from S. lignicolum were studied to elucidate the essential groups for catalysis. The hydrolysis of Z-Phe-Leu-Ala-Ala by acid proteinase A-2 was studied from pH 1 to 5 at 30°C, and the Michaelis constant, Km, and the maximum velocity, V, were found for each pH. The pH-kcat/Km profile was bell-shaped. The pKe1 and pKe2 for the free enzyme were 1.53 and 3.77, respectively. Acid proteinase A-2 does not contain histidihe residues in the molecule. Accordingly, both of the essential groups are considered to be carboxyl residues. Similar experiments were done with acid proteinase C using Suc-Phe-Arg-Ala-Phe-NPhNO2 as substrate. The pKe1 and pKe2 at 30°C were 2.89 and 5.43, respectively. Based on the data at 30°C and 40°C, the heat of dissociation, ΔHe, was calculated by the equation of Van't Hoff. Both of the groups were regarded as carboxyl residues, since the ΔHe were -0.8 and 1.2kcal/mol, respectively.
These results suggest that Scytalidium enzymes have active carboxyl residues participating in the actions, as do the Pepstatin-sensitive acid proteinases.
