2 Running title

In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5'-oxidation. The nicotine ∆5'(1')iminium ion product of this reaction is further metabolized to cotinine by aldehyde oxidase. Previous investigators have reported kinetic parameters for cotinine formation using human liver cytosol as a source of aldehyde oxidase. Using [5-3H]nicotine and radioflow HPLC analysis we determined kinetic parameters for nicotine 5’-oxidation by P450 2A6 and the closely related human extrahepatic P450 2A13 as well as the rodent P450s 2A3, 2A4 and 2A5. The formation of both cotinine and nicotine ∆5'(1')iminium ion was monitored. The Km and Vmax values for P450 2A6 were 144 ± 15 µM and 1.30 ± 0.05 pmol/min/pmol, respectively. Previously reported Km values for cotinine formation by P450 2A6 in the presence of cytosol were much lower, ranging from 11 to 45 µM. P450 2A13 was a somewhat better catalyst of nicotine ∆5'(1')iminium formation, with 2 fold lower Km and 2-fold higher Vmax values than P450 2A6. The rat P450 2A3 and the mouse P450 2A5, which are 85 and 84 % identical to P450 2A6, were much more efficient catalysts of nicotine 5'-oxidation. P450 2A4 was not an efficient catalyst of nicotine metabolism. While 5'-oxidation was the major pathway of nicotine metabolism for al