CYP2A6- and CYP2A13-catalyzed metabolism of the nicotine Δ^5′(1′)iminium ion

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 Δ ^5′(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 Δ^5′(1′)iminium ion, was responsible for generating the inactivating species. In the studies presented here, we confirm that the nicotine Δ^5′(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 Δ^5′(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 Δ ^5′(1′)iminium ion to cotinine and provide evidence that both enzymes catalyze the sequential metabolism of the nicotine Δ^5′(1′)iminium ion. That is, a fraction of the cotinine formed may not be released from the enzyme before further oxidation to 3′-hydroxycotinine.