Purification and some properties of a new metallo carboxypeptidase of Streptomyces griseus K-1.

Abstract

A new carboxypeptidase was isolated from a mixture of proteolytic enzymes of St. griseus K-1 (pronase) and purified about 30-fold by a procedure including CM-cellulose chromatography, D-Arg-CH-Sepharose chromatography, Sephadex G-100 gel filtration, and ST-Sepharose chromatography with a yield of 55%. In these purification procedures, two kinds of substrates, Cbz-Gly-Leu and Bz-Gly-Arg, which are representative substrates of mammalian pancreatic carboxypeptidase A [EC 3.4.12.2] and B [EC 3.4.12.31], respectively, were used to detect the activity. The activities towards the two substrates were eluted at the same position, and the ratio of the activity with Cbz-Gly-Leu to that with Bz-Gly-Arg was always constant. On isoelectric focusing, the purified enzyme protein appeared as a single symmetrical peak at pH 5.2, and the two activities overlapped completely. Metal analysis of the enzyme showed 0.88 zinc atom per mol of the enzyme; its molecular weight determined by the sedimentation equilibrium method was 34,000. In studies on the enzymic properties, such as pH optimum, pH stability, stability at various temperatures, effect of buffer composition, and optimum activity temperature, no difference between the two activities could be observed. Reversible inhibitions of the activities towards Bz-Gly-Leu and Bz-Gly-Lys by reaction products were studied. Lys or Leu showed competitive-type inhibition and the K1 values for the two activities were parallel. The carboxypeptidase activity was tested with reduced and carboxymethylated pancreatic ribonuclease. The enzyme liberated amino acids sequentially, except for Pro. These observations led us to conclude that the carboxypeptidase of St. griseus K-1 has a new substrate specificity which includes the specificities of mammalian carboxypeptidases A and B.