Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid.
Human N-acetyltransferase 1 (NAT1) is a widely distributed enzyme that catalyses the acetylation of arylamine and hydrazine drugs as well as several known carcinogens, and so its levels in the body may have toxicological importance with regard to drug toxicity and cancer risk. Recently, we showed that p-aminobenzoic acid (PABA) was able to down-regulate human NAT1 in cultured cells, but the exact mechanism by which PABA acts remains unclear. In the present study, we investigated the possibility that PABA-induced down-regulation 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-PABA irreversibly inactivates human NAT1 both in cultured cells and cell cytosols in a time- and concentration-dependent manner. Maximal inactivation in cultured cells occurred within 4 hr of treatment, with a concentration of 30 microM reducing activity by 60 +/- 7%. Dialysis studies showed that inactivation was irreversible, and cofactor (acetyl coenzyme A) but not substrate (PABA) completely protected against inactivation, indicating involvement of 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-PABA decreases NAT1 activity by a direct interaction with the enzyme and appears 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 protein content.
Butcher
N J
NJ
Department of Pharmacology, University of Western Australia, Western Australia 6907, Nedlands, Australia. nbutcher@receptor.pharm.uwa.edu.au
Ilett
K F
KF
Minchin
R F
RF
D010129:4-Aminobenzoic Acid / Q000378:metabolism / Q000494:pharmacology*; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019811:Hydroxylamine / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000201:enzymology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019811:Hydroxylamine; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; D010129:4-Aminobenzoic Acid