Chemical modification of hamster arylamine N-acetyltransferase 2 with isozyme-selective and nonselective N-arylbromoacetamido reagents

Haiqing Wang, Zhijun Guo, Gregory M. Vath, Carston R. Wagner, Patrick E. Hanna

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)153-166
Number of pages14
JournalProtein Journal
Volume23
Issue number2
DOIs
StatePublished - 2004

Bibliographical note

Funding Information:
The authors thank Dr. Thomas Krick, Dr. LeeAnn Higgins, and Dr. Sudha Marimanikkuppan of the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, for their assistance and advice. This research was supported in part by U.S. Public Health Service Grant CA55334 from the National Cancer Institute and a Development Grant in Drug Design from the Department of Medicinal Chemistry, University of Minnesota.

Keywords

  • 2-(acetylamino)fluorene
  • Acetanilide
  • Affinity labeling
  • Mass spectrometry (MS)
  • N-acetyltransferases
  • Tandem mass spectrometry (MS/MS)

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