title: CYP2A6- and CYP2A13-catalyzed metabolism of the nicotine Δ5'(1')iminium ion.
abstract: Nicotine, the major addictive agent in tobacco, is metabolized primarily by CYP2A6-catalyzed oxidation. The product of this reaction, 5'-hydroxynicotine, is in equilibrium with the nicotine Δ5'(1')iminium ion and is further metabolized to cotinine. We reported previously that both CYP2A6 and the closely related extrahepatic enzyme CYP2A13 were inactivated during nicotine metabolism; however, inactivation occurred after metabolism was complete. This led to the hypothesis that oxidation of a nicotine metabolite, possibly the nicotine Δ5'(1')iminium ion, was responsible for generating the inactivating species. In the studies presented here, we confirm that the nicotine Δ5'(1')iminium ion is an inactivator of both CYP2A6 and CYP2A13, and inactivation depends on time, concentration, and the presence of NADPH. Inactivation was not reversible and was accompanied by a parallel loss in spectrally active protein, as measured by reduced CO spectra. These data are consistent with the characterization of the nicotine Δ5'(1')iminium ion as a mechanism-based inactivator of both CYP2A13 and CYP2A6. We also confirm that both CYP2A6 and CYP2A13 catalyze the metabolism of the nicotine Δ5'(1')iminium ion to cotinine and provide evidence that both enzymes catalyze the sequential metabolism of the nicotine Δ5'(1')iminium ion. That is, a fraction of the cotinine formed may not be released from the enzyme before further oxidation to 3'-hydroxycotinine.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Masonic Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE, Minneapolis, MN 55455, USA.
authors.forename: Linda B
authors.initials: LB
authors.lastname: von Weymarn
authors.forename: Cassandra
authors.initials: C
authors.lastname: Retzlaff
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Murphy
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003367:Cotinine / Q000031:analogs & derivatives / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009538:Nicotine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D014026:Nicotiana; D014316:Tritium
chemical_list: D003374:Coumarins; D004791:Enzyme Inhibitors; D018722:Nicotinic Agonists; D014316:Tritium; C001381:hydroxycotinine; D009249:NADP; D009538:Nicotine; C034629:nicotine imine; C030123:coumarin; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D003367:Cotinine