Purification and Properties of the Pyrrolidonecarboxylate Peptidase of Streptococcus faecium

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Abstract
Pyrrolidonecarboxylate peptidase (EC 3.4.11.8) from S. faecium was purified by fractionation with streptomycin sulfate and (NH4)2SO4, by chromatography on Sephadex G200 and DEAE-cellulose, and by preparative electrophoresis on Sephadex G25. The purified enzyme on acrylamide gel showed a strong protein band which contained enzyme activity and a very faint band which had no activity. The subunit MW of the purified enzyme was estimated by acrylamide gel electrophoresis in sodium dodecyl sulfate to be 42,000 .+-. 1000. The enzyme showed optimum activity at pH 7.6 and was unstable in the absence of 2-mercaptoethanol. The sensitivity of the enzyme to alkylating agents (N-ethylmaleimide and iodoacetamide) suggested that free SH groups were essential for enzyme activity. The enzyme was rapidly inactivated above 45.degree. C. The values of the Km obtained with various L-pyrrolidonecarboxylyl dipeptides were similar although there was a 10-fold range in the maximal rates of hydrolysis of these substrates. Inhibition studies showed that the substrate analogs 2-pyrrolidone and pyrrolidonecarboxylate are competitive inhibitors of the enzyme. The binding of substrates and inhibitors to the active site of the enzyme is discussed.