By using the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP), a novel polymorphism of CYP2A6, CYP2A6*6, was detected in 0.4% of the Japanese population. To study the enzymatic properties of the CYP2A6.6 protein with a single amino acid substitution of arginine 128 to glutamine, both this isozyme and the CYP2A6.1 protein (wild-type) were produced in insect cells using a baculovirus system. Coumarin 7-hydroxylation, which reflects

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... activity, was significantly reduced (one-eighth of normal) in cell lysate from CYP2A6*6-transfected Sf9 cells compared with that lysate from CYP2A6*1-transfected cells. To clarify the mechanism of inactivation of the CYP2A6.6 enzyme, the heme content and reduced CO difference spectrum were examined. Although CYP2A6.6 retained about onehalf the heme content of CYP2A6.1, the reduced CObound Soret peak was completely lost. These results suggest that the inactivation of CYP2A6.6 is mainly due to disordering of the holoprotein structure rather than a failure of heme incorporation. Cytochrome P450 (CYP) 1 is a superfamily of hemoproteins, many of which can metabolize xenobiotics such as procarcinogens, drugs, and environmental pollutants. CYP2A6 2 is a major hepatic member of the family, which metabolizes pharmaceutical agents such as coumarin and activates some procarcinogens, including 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone and N-nitrosodiethylamine (1, 2). CYP2A6 also