Irreversible Inactivation of Arylamine N-Acetyltransferases in the Presence of N-Hydroxy-4-Acetylaminobiphenyl:  A Comparison of Human and Hamster Enzymes

Abstract

Arylamine N-acetyltransferases (NATs) catalyze the N-acetylation of arylamines, the O-acetylation of N-arylhydroxylamines, and the conversion of N-(aryl)acetohydroxamic acids to N-acetoxyarylamines. NATs also undergo irreversible inactivation in the presence of N-(aryl)acetohydroxamic acids. We previously established that inactivation of hamster NAT1 by N-hydroxy-2-acetylaminofluorene is the result of sulfinamide adduct formation with Cys68. The purpose of this research was to determine the kinetics of inactivation of hamster NAT1, hamster NAT2, and human NAT1 by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), to identify the amino acids that are modified upon NAT-catalyzed bioactivation of N-OH-4-AABP, to characterize the adducts and to identify factors that influence the propensity of NATs to undergo inactivation by N-arylhydroxamic acids. Mass spectrometric analysis of the NATs, after incubation with N-OH-4-AABP, revealed that the principal adduct of each protein was a (4-biphenyl)sulfinamide. Proteolysis of the adducted NATs caused hydrolysis of the sulfinamides to sulfinic acids. Tandem mass spectrometric analysis of the modified peptides revealed that each NAT isozyme contained a sulfinic acid on the Cys68 side chain. Minor adducts were identified as 4-aminobiphenyl conjugates of tyrosines. Hamster NAT1 was more rapidly inactivated by N-OH-4-AABP than either hamster NAT2 or human NAT1, and it was demonstrated that 4-nitrosoobiphenyl, which forms the sulfinamide adducts, accumulates during incubation of N-OH-4-AABP with hamster NAT2 and human NAT1 but not during incubations with hamster NAT1. Steady state kinetic analysis of the hydrolysis of acetylated NATs revealed that the half-lives of acetylated hamster NAT2 and human NAT1 are 7-8-fold greater than that of acetylated hamster NAT1. These results support the proposal that the mechanism of inactivation of NATs by N-OH-4-AABP involves initial deacetylation to produce N-OH-4-aminobiphenyl, which after oxidative conversion to 4-nitrosobiphenyl reacts with Cys68 to form a sulfinamide. The relatively short half-life of the acetylated form of hamster NAT1 contributes to its greater susceptibility to inactivation by N-OH-4-AABP.

Arylamine N-Acetyltransferase 1: A Novel Drug Target in Cancer Development

“…Nitrosoarenes derived from arylamines that were efficiently acetylated by NAT1 were the most potent inactivators of the enzyme. Mass spectrometric analysis of FLAG-tagged NAT1 protein purified from HeLa cells treated with the nitrosoarene 4-nitrosobiphenyl confirmed that intracellular inactivation of NAT1 was due to the formation of a sulfinamide adduct between 4-nitrosobiphenyl and the active-site cysteine (Cys 68 ) , confirming earlier in vitro studies using hamster NAT (Guo et al, 2004;Wang et al, 2005b).…”

Inflammatory cytokines suppress arylamine N-acetyltransferase 1 in cholangiocarcinoma cells

“…The catalytic activity of NAT enzymes is dependent on a reactive cysteine residue, since its activity is inhibited when this residue is modified by N-hydroxy-arylamine compounds, hydroxamic acids [22] , enzyme substrates [10] , as well as oxidant species [12,13,23] . Oxidative stress and chronic inflammation are inseparable such that inflammation inevitably produces oxidant species, resulting in tissue damage [24] .…”

Xenobiotic-Metabolizing Enzymes in Human Lung

“…It was also reported that CYP2A13 has a significant activity in metabolizing aflatoxin B(1) to its carcinogenic/toxic AFB(1)-8,9-epoxide and AFM(1)-8,9-epoxide at both low (15 μM) and high (150 μM) substrate concentrations. Amino acid residues Ala (117) and His(372) in CYP2A13 protein are shown to be important for aflatoxin B 1 epoxidation and its related cytotoxicity, suggesting that CYP2A13-catalyzed metabolic activation in situ may play a critical role in human lung carcinogenesis related to inhalation exposure to aflatoxin B 1 [62].…”

“…Arylamine NATs catalyze the N-acetylation of arylamines, the O-acetylation of N-arylhydroxylamines, and the conversion of N-(aryl)acetohydroxamic acids to N-acetoxyarylamines. NATs also undergo irreversible inactivation in the presence of N-(aryl)acetohydroxamic acids [117]. NAT activity is involved in paclitaxel-induced N-acetylation of 2-aminofluorene [118].…”