Metallocarboxypeptidases: Stability Constants and Enzymatic Characteristics

In a previous communication (l), we have described some of the enzymatic and physicochemical properties of a series of metallocarboxypeptidases prepared from the metal-free apoenzyme of bovine carboxypeptidase A. This paper presents the stability constants and enzymatic activities of a series of metallocarboxypeptidases produced when 1 g atom of zinc in the native enzyme is replaced by 1 g atom of manganese, cobalt, nickel, copper, cadmium, or mercury.Significantly, the substitution of the various metals for zinc in carboxypeptidase induces marked changes in relative activities and specificity of the enzyme.The order and magnitude of the stability constants of this series of metallocarboxypeptidases are in accord with the hypothesis that a sulfur and a nitrogen atom of the apoenzyme bind these metal ions to constitute the active center (2-4).The release of two Hi-ions when a metal atom is bound to apocarboxypeptidase constitutes further experimental support for this hypothesis, since the apparent p/c values of the ionizing groups are consistent with those to be expected of a sulfhydryl and a nitrogenous group of amino acid residues (5). EXPERIMENTAL PROCEDUREBeef Pancreas Carboxypeptiduse [(CPD)Znll-Four times recrystallized zinc carboxypeptidase was prepared from beef pancreas acetone powder2 by the method of Allan et aZ.3The solution of the final crystals in 1.0 M NaCl-0.1 M Tris buffer, pH 7.5, was homogeneous in the ultracentrifuge and by moving boundary electrophoresis when examined in 0.3 ionic strength LiCl buffers of pH 6.6 to 10.5.The proteolytic coefficient, C, of this preparation was 25 to 30 at pH 7.5, 25", and 6 to 7 at pH 7.5, 0".The esterase activity, expressed as a zero order rate constant, k (see below), was 1.15 X lo3 pM H+ per minute per mg of N at pH 7.5, 25".The zinc to protein ratio was 1950 pg per g of protein or 1.03 g atom per mole, assuming a molecular weight of 34,300 for the protein (6, 7).