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 S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X 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 S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.
D000577:Amides / Q000378:metabolism; D001665:Binding Sites; D002176:Candida albicans / Q000201:enzymology*; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D000577:Amides; D010450:Endopeptidases; D009596:Nitrophenols; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D014443:Tyrosine; C028121:N-benzyloxycarbonyltyrosine-4-nitrophenyl ester; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins