Functional characterization of allelic variants of polymorphic human cytochrome P450 2A6 (CYP2A6*5, *7, *8, *18, *19, and *35).

Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic compounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic variants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms. Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in Escherichia coli and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic variant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A helix and the F-G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 allelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation, CYP2A6*8 and *35 displayed increased K(m) values whereas CYP2A6*18 and *19 showed decreased k(cat) values, which resulted in lower catalytic efficiencies (k(cat)/K(m)). In the case of nicotine 5-oxidation, the CYP2A6*19 variant exhibited an increased K(m) value, whereas CYP2A6*18 and *35 showed much greater decreases in k(cat) values. These results suggest that individuals carrying these allelic variants are likely to have different metabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6 can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.