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.

Fig. 1.

Primary pathways of nicotine oxidation.

Fig. 2.

Time- and concentration-dependent inactivation of CYP2A6 (A) and CYP2A13 (B) by the nicotine Δ^5′(1′)iminium ion. Activity remaining refers to coumarin 7-hydroxylation activity measured in a secondary reaction at the indicated times. The Δ^5′(1′)iminium ion concentrations are 0 (●), 50 (○), 75 (▾), 100 (▵), and 200 (■) μM for CYP2A6 and 0 (●), 5 (○), 10 (▾), 25 (▵), 50 (■), and 100 (□) μM for CYP2A13. Values are mean ± S.D. from three independent experiments performed in duplicate. The insets represent the double-reciprocal plot generated from the slopes of the lines at the various concentrations.

Fig. 3.

Partition ratio determination for CYP2A13 with the nicotine Δ^5′(1′)iminium ion. The inactivation was allowed to go to completion, then coumarin 7-hydroxylation activity was determined in a secondary reaction. Values are the means ± S.D. of three independent experiments preformed in duplicate. The arrow indicates the intercept used to determine the partition ratio.

Fig. 4.

Radioflow HPLC analysis of CYP2A13-mediated metabolism of nicotine Δ^5′(1′)iminium ion on system I. CYP2A13 (3 pmol) was incubated with 20 μM [5-³H]nicotine Δ^5′(1′)iminium ion (specific activity, 0.1 μCi/nmol) for 5 min (A) or 20 min (B). The retention times were confirmed by coelution with standards in each sample.

Fig. 5.

Radioflow HPLC analysis of nicotine Δ^5′(1′)iminium ion metabolism by CYP2A6 and CYP2A13 on system II. [5-³H]nicotine Δ^5′(1′)iminium ion (5 μM; 4 μCi/nmol) was incubated with CYP2A13 (25 pmol) for 5 min (A) and 20 min (B) or with CYP2A6 (25 pmol; 4 μCi/nmol) for 5 min (C) and 10 min (D). Retention times were confirmed by coelution with standards. A mix of standards, which included cis-3HCOT (r_t = 17.3 min), trans-3HCOT (r_t = 18.0 min), NHCOT (r_t = 19.2 min), norcotinine (r_t = 21.1 min), 5HCOT (r_t = 21.7 min), nornicotine (r_t = 23.0 min), and cotinine (r_t = 25.1 min), was added to each sample. cis-Nicotine N-oxide (r_t = 9.1 min), trans-nicotine N-oxide (r_t = 10.2 min), myosmine (r_t = 29.2 min), and β-nicotyrine (r_t = 41.1 min) were added to a subset of samples.