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 language | English (US) |
|---|---|
| Pages (from-to) | 631-642 |
| Number of pages | 12 |
| Journal | Protein Journal |
| Volume | 22 |
| Issue number | 7-8 |
| State | Published - Dec 1 2003 |
Keywords
- 2-(Acetylamino)fluorene
- Acetanilide
- Affinity labeling
- Mass spectrometry (MS)
- N-acetyltransferases
- Tandem mass spectrometry (MS/MS)