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Acetyl-Coenzyme A: Arylamine N-Acetyltransferase

ROLE OF THE ACETYL-ENZYME INTERMEDIATE AND THE EFFECTS OF SUBSTITUENTS ON THE RATE

From the ¹ From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154

The reactions of p-nitroacetanilide and p-nitrophenyl acetate with aromatic and aliphatic amines catalyzed by a partially purified preparation of acetyl-coenzyme A:arylamine acetyltransferase follow "ping-pong" kinetics. At a given concentration of p-nitroacetanilide the identical maximum velocities observed for five substituted anilines, semicarbazide, hydroxylamine, and hydrazine, suggest that these reactions proceed through the rate-determining formation of a common acetyl-enzyme intermediate which reacts with the acceptor amine in a fast step. The maximum velocities are directly proportional to the p-nitroacetanilide concentration. p-Nitrophenyl acetate is the most active acyl donor substrate known for the enzyme, with a maximum velocity approximately 140-fold larger than that of acetyl-CoA. With weakly basic anilines the maximum velocity is different for different acyl acceptors, suggesting that with these substrates the reaction of the acetyl-enzyme intermediate with the acceptor amine is rate determining and that the saturation with increasing amine concentration represents binding of the acceptor amine to the acyl-enzyme. With strongly basic anilines the maximum velocities and the K_m for p-nitrophenyl acetate are identical, within experimental error, for all anilines; this is interpreted as evidence for a change to rate-determining acylation of the enzyme with these more reactive nucleophiles. The increase in reactivity with increasing aniline basicity suggests the development of a partial positive charge on the attacking nitrogen atom, which may be accompanied by a relatively small degree of proton removal by a general base catalyst. It is suggested that the wide variation in the sensitivity of enzyme-catalyzed acyl transfer reactions to the basicity of the nucleophile or leaving group reflects different degrees of proton removal or addition by general acid-base catalysts in the transition state.

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