Irreversible inactivation of arylamine N-acetyltransferases in the presence of N-hydroxy-4-acetylaminobiphenyl: A comparison of human and hamster enzymes

Haiqing Wang, Carston R. Wagner, Patrick E. Hanna

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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.

Original languageEnglish (US)
Pages (from-to)183-197
Number of pages15
JournalChemical research in toxicology
Volume18
Issue number2
DOIs
StatePublished - Feb 1 2005

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N-hydroxy-4-acetylaminobiphenyl
Arylamine N-Acetyltransferase
Acetyltransferases
Cricetinae
Enzymes
Sulfinic Acids
Acetylation
Hydrolysis
Proteolysis
2-Acetylaminofluorene
Kinetics
hydroxide ion

Cite this

@article{c249f486a002436a84d5145facd4cedd,
title = "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.",
author = "Haiqing Wang and Wagner, {Carston R.} and Hanna, {Patrick E.}",
year = "2005",
month = "2",
day = "1",
doi = "10.1021/tx049801w",
language = "English (US)",
volume = "18",
pages = "183--197",
journal = "Chemical Research in Toxicology",
issn = "0893-228X",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Irreversible inactivation of arylamine N-acetyltransferases in the presence of N-hydroxy-4-acetylaminobiphenyl

T2 - A comparison of human and hamster enzymes

AU - Wang, Haiqing

AU - Wagner, Carston R.

AU - Hanna, Patrick E.

PY - 2005/2/1

Y1 - 2005/2/1

N2 - 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.

AB - 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.

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U2 - 10.1021/tx049801w

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