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

Research output: Contribution to journalArticle

Abstract

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 NAT1 and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NAT1 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
JournalProtein Journal
Volume22
Issue number7-8
StatePublished - Dec 1 2003

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Keywords

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

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