Six Novel Nonsynonymous CYP1A2 Gene Polymorphisms: Catalytic Activities of the Naturally Occurring Variant Enzymes

  1. Norie Murayama1,
  2. Akiko Soyama1,
  3. Yoshiro Saito,
  4. Yukiko Nakajima,
  5. Kazuo Komamura,
  6. Kazuyuki Ueno,
  7. Shiro Kamakura,
  8. Masafumi Kitakaze,
  9. Hideo Kimura,
  10. Yu-ichi Goto,
  11. Osamu Saitoh,
  12. Masaaki Katoh,
  13. Teiichi Ohnuma,
  14. Mitsuru Kawai,
  15. Kenji Sugai,
  16. Taisuke Ohtsuki,
  17. Chieko Suzuki,
  18. Narihiro Minami,
  19. Shogo Ozawa and
  20. Jun-ichi Sawada
  1. Project Team for Pharmacogenetics (N.Mu., A.S., Y.S., Y.N., S.O., J.-i.S.), Division of Biochemistry and Immunochemistry (Y.S., J.-i.S.), Division of Pharmacology (S.O.), National Institute of Health Sciences, Tokyo, Japan; Departments of Cardiology (K.K., S.K., Ma.Ki.), Cardiovascular Dynamics Research Institute (K.K.), and Pharmacy (K.U.), National Cardiovascular Center, Osaka, Japan; National Institute of Neuroscience (H.K., Y.-i.G., N.Mi.); and National Center Hospital for Mental Nervous and Muscular Disorders (O.S., Ma.Ka., Te.O., Mi.Ka., K.S., Ta.O., C.S., N.Mi.), National Center for Neurology and Psychiatry, Tokyo, Japan
  1. Address correspondence to:
    Dr. Shogo Ozawa, Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. E-mail: sozawa{at}nihs.go.jp

Abstract

Six novel nonsynonymous nucleotide alterations were found in the cytochrome P450 1A2 gene in a Japanese population, which resulted in the following amino acid substitutions: T83M, E168Q, F186L, S212C, G299A, and T438I. These individuals were heterozygous for the amino acid substitutions. The potential functional alterations caused by the amino acid substitutions were characterized by a cDNA-mediated expression system using Chinese hamster V79 cells. Among the six CYP1A2 variants, F186L showed the most profound and statistically significant reduction in O-deethylation of phenacetin and 7-ethoxyresorufin. Kinetic analyses performed for the ethoxyresorufin O-deethylation revealed that the Vmax of the F186L variant was approximately 5% of that of the CYP1A2 wild type, despite a 5-fold lower Km value of the variant, the consequence of which was reduced enzymatic activity toward the substrate. Thus, for the first time, phenylalanine at residue 186 is suggested to be a critical amino acid for catalytic activity.

Footnotes

  • This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ-2, MPJ-3, and MPJ-6) of the Organization for Pharmaceutical Safety and Research of Japan.

  • DOI: 10.1124/jpet.103.055798.

  • ABBREVIATIONS: P450, cytochrome P450; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; SNP, single nucleotide polymorphism; HPLC, high-performance liquid chromatography; SRS, substrate recognition sequence.

  • ↵1 These authors contributed equally to this work.

    • Received June 20, 2003.
    • Accepted October 9, 2003.
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  1. Published online before print October 16, 2003, doi: 10.1124/jpet.103.055798 JPET January 2004 vol. 308 no. 1 300-306
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