title: Bioactivation of N-arylhydroxamic acids by rat hepatic N-acetyltransferase. Detection of multiple enzyme forms by mechanism-based inactivation.
abstract: Enzymatic N,O-acyltransfer of carcinogenic N-arylhydroxamic acids such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) results in the production of reactive electrophiles that can bond covalently with nucleophiles and also can cause inactivation of acyltransferase activity in a mechanism-based manner. Incubation of partially purified rat hepatic N-acetyltransferases (NAT) with N-OH-AAF resulted in extensive inactivation of N-OH-AAF/4-aminoazobenzene (AAB) N,N-acetyltransferase and acetyl coenzyme A (AcCoA)/procainamide (PA) N-acetyltransferase activities, whereas AcCoA/p-aminobenzoic acid (PABA) N-acetyltransferase activity was inhibited only slightly. Affinity chromatography with Sepharose 6B 2-aminofluorene (2-AF) resulted in the separation of two NAT activities. NAT I primarily catalyzed the AcCoA-dependent acetylation of PABA; NAT II catalyzed, N,N-acetyltransfer (N-OH-AAF/AAB), AcCoA/PA N-acetyltransfer and N-OH-AAF N,O-acyltransfer (AHAT) activities. Most of the AcCoA/2-AF N-acetyltransferase activity eluted in the NAT II fraction. Results of inactivation experiments with N-OH-AAF and the NAT II fractions suggested that one NAT isozyme was responsible for catalyzing the N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransfer reactions and that inactivation of NAT II correlated with the extent of covalent binding to protein. Further purification of the NAT II fractions by chromatofocusing resulted in a 1300-fold purification of the N-OH-AAF/AAB activity and the coelution of N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransferase activities. These studies indicate that N,O-acyltransfer, arylhydroxamic acid-dependent N-acetylation of arylamines (N,N-acetyltransfer), and AcCoA-dependent N-acetylation of PA may be catalyzed by a common enzyme in rat liver, whereas a second enzyme is responsible for the AcCoA-dependent N-acetylation of PABA.
authors.affiliation: Department of Pharmacology, University of Minnesota, Minneapolis 55455.
authors.forename: M J
authors.initials: MJ
authors.lastname: Wick
authors.forename: P E
authors.initials: PE
authors.lastname: Hanna
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001711:Biotransformation; D004789:Enzyme Activation / Q000187:drug effects; D006877:Hydroxamic Acids / Q000493:pharmacokinetics*; D006881:Hydroxyacetylaminofluorene / Q000378:metabolism / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009596:Nitrophenols / Q000494:pharmacology; D010261:Paraoxon / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D013379:Substrate Specificity
chemical_list: D006877:Hydroxamic Acids; D007527:Isoenzymes; D009596:Nitrophenols; D006881:Hydroxyacetylaminofluorene; C002887:bis(4-nitrophenyl)phosphate; D000123:Acetyltransferases; D001191:Arylamine N-Acetyltransferase; D010261:Paraoxon