THE PURIFICATION AND PROPERTIES OF YEAST PROTEINASE-B FROM CANDIDA-ALBICANS

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30 000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and .**GRAPHIC**. The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat/Km 3536000 M-1 .cntdot. s-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat/Km 2250000 M-1 .male. s-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat/Km 1000000 M-1 .cntdot. s-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite .**GRAPHIC**. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat/Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825000 M-1 .cntdot. s-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333 000 M-1 .cntdot. s-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.