Mass spectrometric investigation of the mechanism of inactivation of hamster arylamine N-acetyltransferase 1 by N-hydroxy-2-acetylaminofluorene.

Arylamine N-acetyltransferases (NATs) are expressed in most mammalian tissues. NATs catalyze the N-acetylation of primary arylamines, the O-acetylation of N-arylhydroxylamines, and the N,O-transacetylation of N-arylhydroxamic acids. The latter two reactions result in formation of reactive, electrophilic N-acetoxyarylamines, which are considered to be the ultimate carcinogenic metabolites of certain environmental and dietary arylamines. Incubation of various N-(aryl)acetohydroxamic acids, such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) with hamster NAT1, results in time-dependent, concentration-dependent, and kinetically first-order irreversible inactivation of the enzyme. N-OH-AAF also causes in vivo inactivation of NAT1. The purpose of this research was to investigate the molecular mechanism of NAT1 inactivation by identifying the amino acid residues that undergo covalent modification upon NAT1-catalyzed bioactivation of N-OH-AAF and by characterizing the chemical structures of the adducts. Electrospray ionization quadrupole time-of-flight mass spectrometric analysis of NAT1 that had been incubated with N-OH-AAF revealed that the mass of the major adduct (+195 Da) was consistent with a (2-fluorenyl)sulfinamide modification. The major adduct underwent hydrolysis to yield a protein with a molecular mass that corresponded to a sulfinic acid-modified NAT1. Treatment of NAT1 with 2-nitrosofluorene resulted in a modification (+195 Da) that was identical in mass to that obtained with N-OH-AAF-inactivated enzyme. Matrix-assisted laser desorption-ionization quadrupole time-of flight tandem mass spectrometric (MALDI Q-TOF MS/MS) analysis revealed that the modified residue was the catalytically essential Cys68. MALDI Q-TOF MS/MS sequencing of peptides from protease digests of inactivated NAT1 also identified two minor adducts at Tyr17 and Tyr186, each of which was covalently conjugated with 2-aminofluorene. Thus, the mechanism of inactivation of NAT1 by N-OH-AAF involves NAT1-catalyzed deacetylation to afford N-hydroxy-2-aminofluorene, which after oxidative conversion to 2-nitrosofluorene, forms a sulfinamide adduct by reacting with Cys68. GSH had little effect on the inactivation of NAT1 by N-OH-AAF, although high concentrations of cysteine attenuated both the extent of inactivation and the sulfinamide adduct formation.