Phosphoenolpyruvate carboxytransphosphorylase from propionic acid bacteria requires a freely dissociable divalent metal such as Mg2+ but is inhibited by a variety of structurally unrelated metal chelators even in the presence of a large excess of Mg2+. The results indicate that the enzyme has a requirement for two types of metal cations, one dissociable (type I) and one tightly bound (type II). In addition a third metal (a heavy metal) is stimulatory for the pyruvate reaction. Both the oxalacetate and pyruvate reactions are inhibited to the same degree by almost identical concentrations of these chelators suggesting that the type II metal is required for both reactions. No evidence was found of a requirement for a specific monovalent ion by carboxytransphosphorylase. EDTA-treated and dialyzed enzyme is inactive with Mg2+ alone but addition of Co2+ along with Mg2+ activates the enzyme for the oxalacetate reaction and Cu2+ is effective for the pyruvate reaction. 60Co2+ binding experiments proved that the reactivation was not due to binding of 60Co2- to an apoenzyme. Tests of EDTA-treated and dialyzed enzyme in combination with untreated enzyme showed that the EDTA-treated enzyme was inhibitory apparently because it retained bound EDTA which inhibited the untreated enzyme. It is proposed that Co2+ activates by removing the EDTA from the type II metal. The inhibition of the oxalacetate reaction by o-phenanthroline is shown to be of two types, an instantaneous inhibition which is reversible and a time-dependent inhibition which is irreversible; there is little evidence for the time-dependent, irreversible inhibition of the pyruvate reaction. These results have been interpreted with reference to and appear consistent with the present hypothesis that two enzyme forms of carboxytransphosphorylase exist, one catalyzing the oxalacetate reaction and the other, containing a heavy metal, catalyzing the pyruvate reaction. © 1969, American Chemical Society. All rights reserved.
