Arylamine N-Acetyltransferases: Covalent Modification and Inactivation of Hamster NAT1 by Bromoacetamido Derivatives of Aniline and 2-Aminofluorene

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

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Kinetic analysis of the inactiviation of hamster NAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an active site directed affinity label whereas bromoacetanilide acts as a bimolecular alkylating agent. ESI MS analysis of NAT1 treated with Br-AAF showed that a single molecule of 2-acetylaminofluorene had been incorporated. Proteolysis with pepsin followed by sequencing of adducted peptides by ESI MS/ MS identified the modified residue as the catalytically essential Cys-68. ESI Q-TOF MS analysis of NAT1 that had been treated with bromoacetanilide resulted in identification of a monoadducted protein as the primary product and a diadducted protein as a minor product. Pepsin digestion of bromoacetanilide-inactivated NATI and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NATI led to the identification of a second modified peptide which was adducted at Cys-44. The data reveal substantial differences in the interactions of small hydrophobic alkylating reagents with hamster NAT1.

Original languageEnglish (US)
Pages (from-to)631-642
Number of pages12
JournalJournal of Protein Chemistry
Issue number7-8
StatePublished - Nov 1 2003



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

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