Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamineof p-aminobenzoic acid

Human N-acetyltransferase 1 (NAT1) is a widely distributed enzyme that cata lyses the acetylation of arylamine and hydrazine drugs as well as several k nown carcinogens, and so its levels in the body may have toxicological impo rtance with regard to drug toxicity and cancer risk. Recently, we showed th at p-aminobenzoic acid (PABA) was able to down-regulate human NAT1 in cultu red cells, but the exact mechanism by which PABA acts remains unclear. In t he present study, we investigated the possibility that PABA-induced down-re gulation involves its metabolism to N-OH-PABA, since N-OH-AAF functions as an irreversible inhibitor of hamster and rat NAT1. We show here that N-OH-P ABA irreversibly inactivates human NAT1 both in cultured cells and cell cyt osols in a time- and concentration-dependent manner. Maximal inactivation i n cultured cells occurred within 4 hr of treatment, with a concentration of 30 muM reducing activity by 60 +/- 7%. Dialysis studies showed that inacti vation was irreversible, and cofactor (acetyl coenzyme A) but not substrate (PABA) completely protected against inactivation, indicating involvement o f the cofactor-binding site. In agreement with these data, kinetic studies revealed a 4-fold increase in cofactor K-m, but no change in substrate K-m for N-OH-PABA-treated cytosols compared to control. We conclude that N-OH-P ABA decreases NAT1 activity by a direct interaction with the enzyme and app ears to be a result of covalent modification at the cofactor-binding site. This is in contrast to our findings for PABA, which appears to reduce NAT1 activity by down-regulating the enzyme, leading to a decrease in NAT1 prote in content. BIOCHEM PHARMACOL 60;12: 1829-1836, 2000. (C) 2000 Elsevier Sci ence Inc.