Purification and properties of three endopeptidases from baker's yeast

Abstract

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45 000 and 54 000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7–3.2 and casein at pH 2.4–2.8 and 5.5–6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4–6 M urea. Urea also stimulated the enzyme activity by 30–50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X–Tyr and X–Phe type. A proteinase B (PRB) preparation of approximately Mr 33 000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at −20 °C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72 000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15–Tyr16 peptide bond of the oxidized β-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at −20 °C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB. Similar to PRA, 4 M urea shifted its pH optimum for hydrolysis of protein substrates. PRY degraded apo-aminopeptidase Y much more efficiently than PRB or a PRA–PRB mixture. The possibility of PRY being a precursor form of PRA and PRB is discussed.Key words: yeast, endopeptidase, proteinase, purification.