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1996 Fiscal Year Final Research Report Summary

MECHANISM FOR OXYGEN ACTIVATION BY CYTOCHROME P450cam

Research Project

Project/Area Number 07458163
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionSCHOOL OF MEDICINE,KEIO UNIVERSITY

Principal Investigator

ISHIMURA Yuzuru  DEPARTMENT OF BIOCHEMISTRY,KEIO UNIVERSITY,PROFESSOR, 医学部, 教授 (40025599)

Co-Investigator(Kenkyū-buntansha) NAGANO Shingo  DEPARTMENT OF BIOCHEMISTRY,KEIO UNIVERSITY,INSTRUCTOR, 医学部, 助手 (60286440)
MIKI Hideho  DEPARTMENT OF BIOCHEMISTRY,KEIO UNIVERSITY,INSTRUCTOR, 医学部, 助手 (90229667)
HIROSE Tadaaki  PHARMACEUTICAL INSTITUTE.KEIO UNIVERSITY,ASSIST PROF., 医学部, 講師 (60051405)
EGAWA Tsuyoshi  DEPARTMENT OF BIOCHEMISTRY,KEIO UNIVERSITY,INSTRUCTOR, 医学部, 助手 (10232935)
SHIMADA Hideo  DEPARTMENT OF BIOCHEMISTRY,KEIO UNIVERSITY,ASSOC.PROF., 医学部, 助教授 (80095611)
Project Period (FY) 1995 – 1996
KeywordsCytochome P450 / P450cam / Activation of molecular oxygen / Acid-base catalyst / Site-directed mutagenesis / Artificial amino acids / Dioxygen-bond cleavage / Threonine / Hydrogen bonding
Research Abstract

Cytochrome P450cam is the terminal mono-oxygenase in the d-camphor hydroxylating system of Pseudomonas putida. Recently the x-ray crystal structure of this enzyme has been solved by Poulos and his associates enabling us to study its exact structure-function. relationship. The enzyme catalyzes the reaction indicated below.
d-Camphor+NADH+H^++O_2*5-exo-hydroxycamphor+NAD^++H_2O
Our previous study with conventional site-directed mutagenesis of the enzyme revealed that a hydroxy (OH) group of Thr252 at the oxygen pocket of the cytochrome plays an important role in the mono-oxygenase reaction : In the absence of the OH group, oxygen consumption and the hydroxylation of d-camphor was uncoupled (Proc.Natl.Acad.Sci.U.S.A.86,7823-7827). Based on this and other evidence, the role of Thr252 has been proposed by us to form a hydrogen bonding net work which is formed this threonine and carboxyl group of Asp251. Such a hydrogen-bonding net work is required for the activation of heme-bound dioxygen (O_ … More 2), i.e.reductive cleavage of the O-O bond in O_2, in the above reaction.
to investigate further the role of the OH group of Thr252 in cytochrome P450cam, artificial amino acids with oxygen-, sulfur-, or nitrogen-containing groups were introduced to the 252 position (in place of OH group of Thr) by the aid of in vitro translation system. Measurrements of the enzyme activity of such mutants showed that only those containing oxygen atoms at that position (-OCH_3, COOCH_3) in place of the OH group retained a large portion of the mono-oxygenase activity (>75% formation of hydroxylated product per O_2 consumed). On the other hand, mutants containing a residue with sulfur or nitrogen (-SCH_3, -NH_3^+) had lost most of the activity (<10% formation of hydroxylated product per O_2 consumed). Thus oxygen atom at the residue of 252 position is important for the mono-oxygenase activity.
On the basis of these results, we propose that the property of the oxygen atom in Thr252 as an acceptor of a hydrogen bond is essential for the formation of a hydrogen-bonding network for proton transfer necessary for the mono-oxygenase reaction described above. Less

  • Research Products

    (19 results)

All Other

All Publications (19 results)

  • [Publications] Kimata Y.,Shimada H.,Hirose T.,and Ishimura Y.: "Role of Thr-252 in cytochrome P450cam:A study with unnatural amino acid mutagenesis" Biochem.Biophys.Res.Commun.208. 96-102 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mukai K.,Mitani F.,Shimada H.and Ishimura Y.: "Involvement of an AP-1 complex in zone-specific expression of the CYP11B1 gene in the rat adrenal cortex" Mol.Cell.Biol.15. 6003-6012 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 牧野龍、島田秀夫: "部位特異的変異体を用いたシトクロムP450反応機構の解析" 生物物理. 35. 1-6 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Unno M.,Ishimura Y.他3名: "Role of Arg112 of cytochrome P450cam in the electron transfer from reduced putidaredoxin." J.Biol.Chem.271. 17869-17874 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Goda N.,Ishimura Y.他5名: "Modulation of mitochondrion-mediated oxidative stress by nitric oxide." Am.J.Physiol.271. H1893-H1899 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mitani F.,Ishimura Y.他3名: "Expression of cytochromes P450aldo and P45011 in rat adrenal gland." Steroids. 62. 57-61 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shimada H.et al.: "Oxygenases and Model Systems" Kluwer Academic Publishers(Editor,T.Funabiki), 195-211(393) (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 島田秀夫、石村巽: "現代化学 増刊27:ヘムタンパク質-構造と機能(分担執筆)" 東京化学同人(菊池吾郎他ポルフィリン研究会編), 105-115(296) (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kimata, Y., Shimada, H., Hirose, T., and Ishimura.Y.: "Role of Thr-252 in cytochrome P450cam : A study with unnatural amino acid mutagenesis." Biochem.Biophys.Res.Commun.208. 96-102 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mitani, F., Ogishima, T., Miyamoto, H.and Ishimuram, Y.: "Localization of P450aldo and P45011b in normal and regenerating rat adrenal cortex." Endocr.Res.21. 413-423 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Suematsu, M., Suzuki, H., Tamatani, T., Iigou, Y., Miyasaka, M., Kannagi, R., Ishimura, Y., Zweitach, B.W., and Schmid-Schonbein, G.W.: "Impairment of selectin-mediated leukocyte adhesion to venular endothelium in spontaneously hypertensive rats." J.Clin.Invest.96. 2009-2016 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tamatani, T., Suematsu, M., Tezuka, K., Hanzawa, N., Tsuji, T., Ishimura, Y., Kannagi, R., Toyoshima, S., and Homma, M.: "Recognition of a consensus carbohydrate structure in the ligands for selectins (CD62s) by a novel antibody against sialyl lewis X in vitro and in vivo." Am.J.Physiol.269. H1282-1287 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Suematsu, M., Goda, N., Sano, T., Kashiwagi, S., Egawa, T., Shinoda, Y., and Ishimura, Y.: "Carbon monoxide : An endogenous modulator of sinusoidal tone in the perfused rat liver." J.Clin.Invest.96. 2431-2437 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mukai, K., Mitani, F., Shimada, H.and Ishimura, Y.: "Involvement of an AP-1 complex in zone-specific expression of the CYP11B1 gene in the rat adrenal cortex." Mol.Cell.Biol.15. G6003-6012 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamaguchi, T., Wakabayashi, Y., Tanaka, M., Sano, T., Ishikawa, H., Nakajima, H., Suematsu, M.and Ishimura, Y.: "Taurocholate induces directional excretion of bilirubin into bile in perfused rat liver." Am.J.Physiol.270. 1028-1032 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Unno, M., Shimada, H., Toda, Y., Makino, R.and Ishimura, Y.: "Role of Arg112 of cytochrome P450cam in the electron transfer from reduced putidaredoxin : Analyzes with site-directed mutants." J.Biol.Chem.271. 17869-17874 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Gada, N., Suematsu, M., Mukai, M., Kiyokawa, K., Natori, M., Nozawa, S.and Ishimura, Y.: "Modulation of mitochondrion-mediated oxidative stress by nitric oxide in human placental trophoblastic cell." Am.J.Physiol.271. H1893-H1899 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sano, T., Shiomi, M., Wakabayashi, Y., Shinoda, Y., Yamaguchi, T., Nimura, Y., Ishimura, Y.and Suematsu, M.: "Endogenous carbon monoxide suppression stimulates bile acid-dependent billiary transport in perfused rat liver." Am.J.Physiol. (Gastrointest.& Liver Physiol.). (in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Iigou, M., Suematsu, M., Higashida, T., Oheda, J., Matsumoto, K., Wakabayashi, Y., Ishimura, Y., Miyasaka, M., and Takashi, T.: "In vivo constitutive expression of ICAM-1 in the rat microcirculation." Am.J.Physiol. (Heart and Circ.Physiol.). (in press). (1997)

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      「研究成果報告書概要(欧文)」より

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Published: 1999-03-09  

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