Acetyl Coenzyme A:Arylamine Acetyltransferase

Abstract

Abstract A preparation of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver is rapidly and completely inactivated by iodoacetamide with a second order rate constant of 1950 m-1 min-1 at 25°, pH 7.5. This inactivation is prevented by p-nitrophenyl acetate, an active substrate, at concentrations as low as 1.7 x 10-8 m, far below the Ks value of 8.6 x 10-3 m. The protection afforded by 1.67 x 10-3 m p-nitrophenyl acetate is removed to varying extents in the presence of acceptor anilines under conditions in which the enzyme is catalyzing acetyl transfer. Saturating concentrations of p-toluidine bring about complete removal of protection, whereas saturating concentrations of p-cyanoaniline bring about only a 19% removal of protection. This effect of anilines exhibits the same dependence upon aniline concentration as does the rate of the normal catalyzed reaction. It is concluded: (a) there is a kinetically significant acetyl-enzyme intermediate that is not subject to inactivation by iodoacetamide; (b) the acetyl-enzyme intermediate is almost certainly a thiol ester; (c) the rate of inactivation by iodoacetamide provides a probe for the fraction of enzyme that exists as acetyl-enzyme during the steady state turnover of substrates; (d) with p-nitrophenyl acetate and p-toluidine acetyl-enzyme formation is the rate-determining step, whereas with the less reactive acceptor p-cyanoaniline the deacylation step is rate-determining, confirming earlier conclusions from kinetic data.