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 journalArticle

7 Citations (Scopus)

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 - Dec 1 2004

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Arylamine N-Acetyltransferase
Alkylation
Chemical modification
Cricetinae
Isoenzymes
Acetylation
Mass spectrometry
Enzymes
Electrospray ionization
Peptides
Amino acids
Labels
Affinity Labels
2-Acetylaminofluorene
Molecules
Biotransformation
Tandem Mass Spectrometry
2-(bromoacetylamino)fluorene
Mass Spectrometry
Amino Acids

Keywords

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

Cite this

Chemical modification of hamster arylamine N-acetyltransferase 2 with isozyme-selective and nonselective N-arylbromoacetamido reagents. / Wang, Haiqing; Guo, Zhijun; Vath, Gregory M.; Wagner, Carston R; Hanna, Patrick E.

In: Protein Journal, Vol. 23, No. 2, 01.12.2004, p. 153-166.

Research output: Contribution to journalArticle

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