Studies on hamster recombinant arylamine N-acetyltransferases: Part I. Covalent modification and inactivation of rNAT1 by bromoacetamido derivatives of aniline and 2-aminofluorene. Part II. Mass spectrometric investigation of the inactivation of rNAT1 by

Arylamine N-acetyltransferases (NATs) are phase II enzymes that catalyze the AcCoA dependent N-acetylation of primary arylamines, the O-acetylation of N-arylhydroxylamines and the N,O-transacetylation of N-arylhydroxarnic acids. The latter two reactions generate electrophilic N-acetoxy esters of N-arylhydroxylamines, which react with DNA and proteins. The interactions of hamster rNAT1 with potential affinity labels and the mechanism of hamster rNAT1mediated bioactivation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were studied.; Kinetic analysis of the inactivation of rNAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide indicated that Br-AAF acts as an affinity label with a K₁ of 18.9 μM whereas bromoacetanilide behaves as an alkylating agent with a second order inactivation rate constant of 7.3 M−1 sec−1. Electrospray ionization mass spectrometric (ESI MS) analysis of the Br-AAF-treated rNAT1 revealed that only one protein adduct was formed. Electrospray ionization quadrupole time-of-flight mass spectrometric (ESI-Q-OF MS) analysis of bromoacetanilide-treated rNAT1 revealed two protein adducts. Restricted proteolysis of the adducted protein and subsequent electrospray ionization tandem mass spectrometric (ESI MS/MS) sequencing of the resulting adducted peptide indicated that both compounds modified the catalytically essential Cys68. In addition, Cys44 was found to be modified by bromoacetanilide.; Certain N-arylhydroxamic acids, such as N-OH-AAF, irreversibly inactivate hamster rNAT1. ESI-Q-TOF MS analysis of N-OH-AAF-treated rNAT1 revealed a major adduct (+195 Da) corresponding to a (2-fluorenyl)-sulfinamide modification and a minor adduct (+179 Da) corresponding to the adduction of 2-aminofluorene. The major adduct (+195 Da) underwent hydrolysis to yield an rNAT 1 with a cysteine sulfinic acid residue upon exposure to 2% formate during HPLC. Treatment of rNAT1 with 2-nitrosofluorene resulted in a modification (+195 Da) that was identical in mass to that obtained with N-OH-AAF-inactivated enzyme. Restricted proteolysis of the adducted protein and subsequent matrix assisted laser desorption ionization quadrupole time-of-flight tandem mass spectrometric (MALDI Q-TOF MS/MS) analysis of the adducted peptide indicated Cys68 as the site of (2-fluorenyl)-sulfinamide modification. Through similar procedures, Tyr17 and Tyr186 were found to be covalently conjugated with 2-aminofluorene (+179 Da). Therefore, the inactivation of rNAT1 by N-OH-AAF is a result of reaction of the catalytically essential Cys68 with 2-nitrosofluorene, which is the oxidative product of N-hydroxy-2-aminofluorene resulting from rNAT1-catalyzed deacetylation of N-OH-AAF.