Chemical modification of hamster arylamine N-acetyltransferase 2 with isozyme-selective and nonselective N-arylbromoacetamido reagents.
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.
Wang
Haiqing
H
Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street S.E., Minneapolis, MN 55455, USA.
Guo
Zhijun
Z
Vath
Gregory M
GM
Wagner
Carston R
CR
Hanna
Patrick E
PE
D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000083:Acetanilides / Q000494:pharmacology; D000107:Acetylation; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003553:Cystine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D000083:Acetanilides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C056005:bromoacetanilide; C075748:2-(bromoacetylamino)fluorene; D003553:Cystine; D015073:2-Acetylaminofluorene; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1