CYP2A6*6, a novel polymorphism in cytochrome p450 2A6, has a single amino acid substitution (R128Q) that inactivates enzymatic activity.
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 CYP2A6 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 one-half the heme content of CYP2A6.1, the reduced CO-bound 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.
Kitagawa
K
K
Department of Environmental Health, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
Kunugita
N
N
Kitagawa
M
M
Kawamoto
T
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D005260:Female; D015894:Genome, Human; D006801:Humans; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011110:Polymorphism, Genetic
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6