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Studies of the Acetyl Coenzyme A Synthetase Reaction

IV. THE REQUIREMENT FOR MONOVALENT CATIONS

From the ¹ From the Department of Pharmacology, Western Reserve University, School of Medicine, Cleveland, Ohio 44106

The monovalent cation requirement for a highly purified acetyl coenzyme A synthetase derived from bovine heart mitochondria was investigated. When removal of stimulatory cations from the reaction components was accomplished, no catalytic activity was detected. Adding chloride salts of rubidium, ammonium, potassium, tris(hydroxymethyl)aminomethane, sodium, or lithium to a final concentration of 5 to 10 mm stimulated the over-all reaction maximally. Stimulating monovalent cations acted catalytically and all of their apparent K_m values fell into a range of 1 to 3 mm.

Activating monovalent cations could be separated into two groups on the basis of their kinetic behavior in the over-all reaction. Cations in the first group (Rb⁺, NH₄⁺, K⁺) gave optimal rates of acetyl coenzyme A formation at concentrations of 10 to 200 mm, showed atypical kinetics at elevated concentrations of acetate (10 to 40 mm), but failed to show altered K_m values in the presence of increasing magnesium ion concentrations (3 to 27 mm). In contrast, cations in the second group (Na⁺ and Li⁺) inhibited the reaction at concentrations greater than 10 mm, had this inhibition partially reversed by monovalent cations in the first group, did not have their kinetics effected by acetate, but did show decreasing K_m values as the magnesium ion concentration was increased from 3 to 27 mm.

Two different activating cations, each at a concentration of 60% of its apparent K_m, produced an appreciably greater reaction rate than a single cation at twice this concentration. Yet the maximal velocity obtainable with a single species was not exceeded by various combinations of activating alkali metal ions.

Activating monovalent cations were also shown to be necessary for formation of enzyme-bound acetyladenylate, either from acetate and adenosine triphosphate or from acetyl coenzyme A and adenosine monophosphate. Substrate quantities of enzyme were used to demonstrate this product of both partial reactions under equilibrium conditions. Optimal yields of acetyladenylate were obtained with 5 mm rubidium or sodium chloride. Sodium chloride at 10 mm decreased this yield, whereas rubidium ions did not.

These observations suggest that activating monovalent cations participate in the catalytic mechanism of acetyl coenzyme A synthetase.

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