1996 Fiscal Year Final Research Report Summary
MECHANISM FOR OXYGEN ACTIVATION BY CYTOCHROME P450cam
| Project/Area Number |
07458163
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | SCHOOL OF MEDICINE,KEIO UNIVERSITY |
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Principal Investigator |
ISHIMURA Yuzuru DEPARTMENT OF BIOCHEMISTRY,KEIO UNIVERSITY,PROFESSOR, 医学部, 教授 (40025599)
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| 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)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Cytochome P450 / P450cam / Activation of molecular oxygen / Acid-base catalyst / Site-directed mutagenesis / Artificial amino acids / Dioxygen-bond cleavage / Threonine / Hydrogen bonding |
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| 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
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Research Products
(19 results)
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[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
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[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
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[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
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[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
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[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
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[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
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[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)
Description
「研究成果報告書概要(欧文)」より
