Chemical Modification of Hamster Arylamine N-Acetyltransferase 2 with Isozyme-Selective and Nonselective N-Arylbromoacetamido Reagents |
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Authors: | Haiqing Wang Zhijun Guo Gregory M Vath Carston R Wagner Patrick E Hanna |
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Institution: | Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street S.E., Minneapolis, MN 55455, USA. |
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Abstract: | Arylamine N-acetyltransferases (NATs) catalyze a variety of biotransformation reactions, including N-acetylation of arylamines and O-acetylation of arylhydroxylamines. Chemical modification of hamster recombinant NAT2 with 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an affinity label for the enzyme whereas bromoacetanilide inactivates NAT2 through a bimolecular alkylation process. Electrospray ionization quadrupole time-of-flight mass spectrometry analysis of Br-AAF-treated NAT2 showed that a single molecule of 2-acetylaminofluorene had been adducted. Peptide sequencing with tandem mass spectrometry identified the catalytically essential Cys68 as the alkylated amino acid. Br-AAF exhibits similar affinity for hamster NAT1 and NAT2, but is a more effective inactivator of NAT1 because, subsequent to the formation of a reversible enzyme-Br-AAF complex, the rate of alkylation of NAT1 is greater than the rate of alkylation of NAT2. Bromoacetanilide alkylates Cys68 and, to a lesser extent, Cys237 of NAT2; it does not exhibit significant selectivity for either NAT1 or NAT2. |
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Keywords: | Acetanilide 2-(acetylamino)fluorene affinity labeling mass spectrometry (MS) N-acetyltransferases tandem mass spectrometry (MS/MS) |
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