Arylamine N-acetyltransferases: covalent modification and inactivation of hamster NAT1 by bromoacetamido derivatives of aniline and 2-aminofluorene.
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.
Guo
Zhijun
Z
Department of Medicinal Chemistry, University of Minnesota, 308 Harvard St. S.E., Minneapolis, MN 55455, USA.
Vath
Gregory M
GM
Wagner
Carston R
CR
Hanna
Patrick E
PE
D015073:2-Acetylaminofluorene / Q000737:chemistry / Q000494:pharmacology*; D000083:Acetanilides / Q000737:chemistry / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003553:Cystine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005449:Fluorenes / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010434:Pepsin A; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors
D000083:Acetanilides; D004791:Enzyme Inhibitors; D005449:Fluorenes; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C056005:bromoacetanilide; C012177:2-aminofluorene; D003553:Cystine; D015073:2-Acetylaminofluorene; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; D010434:Pepsin A