Studies of the Acetyl Coenzyme A Synthetase Reaction: V. THE REQUIREMENT FOR MONOVALENT AND DIVALENT CATIONS IN PARTIAL REACTIONS INVOLVING ENZYME-BOUND ACETYL ADENYLATE

Abstract

Abstract The requirement for monovalent and divalent cations was studied in partial reactions of acetyl coenzyme A synthetase involving enzyme-bound acetyl adenylate; enzyme-bound complexes were isolated by gel filtration on Sephadex G-100. Formation of enzyme-bound acetyl-14C adenylate under equilibrium conditions, either from acetate-14C and magnesium-adenosine triphosphate or from acetyl-14C-CoA and adenosine monophosphate, required addition of activating monovalent cations, e.g. Rb+, Na+, or K+, and certain divalent metal ions, e.g. Ni2+, Fe2+, Cd2++, or Cu2+. The same dependence was shown for the enzymatic reaction of chemically synthesized acetyl adenylate with coenzyme A or magnesium-pyrophosphate. The maximum ratio attained for millimicromoles of enzyme-bound acetyl adenylate formed to units of acetyl-CoA synthetase added was 1.55; approximately the same ratio was obtained when the enzyme was saturated with 63Ni2+. This is compatible with two binding sites for acetyl adenylate and Ni2+ per enzyme molecule. Binding of free acetyl-14C adenylate to the enzyme required divalent cations, and the stoichiometry of divalent metal ion bound (Ni2+) to enzyme-bound adenylate was 1:1. Acetyl adenylate prevented removal of approximately 30% of the enzyme-attached 63Ni2+ by Sephadex, but little decrease in enzyme-associated radioactivity was noted after dialysis of enzyme-bound 63Ni2+ against NiCl. Activating monovalent cations (Rb+ or Na+) were not needed for binding acetyl-14C adenylate to the enzyme, but were required for its formation from substrates or for its reaction with coenzyme A. Any enzyme-bound intermediate formed in the presence of suboptimal concentrations of activating monovalent cations reacted completely with coenzyme A, indicating that formation of the complex, rather than its reaction with acceptor, is probably rate-limiting in the over-all reaction. Activating monovalent cations had a direct effect on enzyme-bound acetyl-14C adenylate as judged by the stabilization of the complex at 4°. Rough estimates of equilibrium constants for formation of enzyme-bound acetyl-14C adenylate from acetate-14C and magnesium-adenosine triphosphate at 4° were 8.7 x 104 m-1 at pH 7.4 and 5.5 x 104 m-1 at pH 8.0; these correspond, respectively to -ΔF°' values of 6.3 and 6.0 kcal per mole. From acetyl-14C-CoA and adenosine monophosphate, comparable constants were 1.9 to 3.0 x 104 m-1 at pH 7.4 and 1.1 x 105 m-1 at pH 8.0; these are equivalent to -ΔF°' values of 6.8 and 6.4 kcal per mole.