1991 Fiscal Year Annual Research Report

多核NMR分光法によるフラビン酵素反応機構の研究

Research Project

Project/Area Number	01480525
Research Institution	Kansai Medical University
Principal Investigator	三浦洌関西医科大学, 医学部, 教授 (70093466)
Co-Investigator(Kenkyū-buntansha)	黒田記代関西医科大学, 医学部, 助手 (30131436) 藤井茂関西医科大学, 医学部, 助教授 (60144482)
Keywords	フラビン酵素 / 反応機構 / 電子移動 / 核磁気共鳴 / NMR / 多核NMR / 安定同位体標識
Research Abstract	D・アミノ酸酸化酵素(DAOと略) DAOによる基質の酸化の機構を調べるために、基質アナログとして、oー,mー,Pーフッ素化安息香酸(それぞれ、OFB,MFB,PFBと略)を用い、これらのアナログのF(19)ーNMR信号を指標にFBとDAOとの結合状態を調べた。その結果、3個のアナログのうち、OFBのF(19)ー信号が他に比べてもっとも大きくシフトし、MFB,PFBの順に小さくなった。このことは、基質Dーアミノ酸がDAOによって酸化される際、アミノ基からフラビンへ電子が移動することを示唆している。 NADPHーアドレノドキシン還元酵素(ADRと略) ADRをC(13)で選択的に標識したFADで再構成し、遊離の状態および、NADP結合状態でC(13)ーNMRを測定した。C(13)標識位置は、フランビン環の2,4,4a,10aー位である。NADP結合状態では、4aーC(13)信号が低磁場シフトしており、NADPHからADRに電子が伝達される時、NADPHがADRに結合することにより、フラビン部分の4aー位の電子密度が下り、NADPHからフラビンへの電子伝達が促進されていることが示唆された。旧黄色酵素(OYEと略) 8ーフルオローFMM再構成したOYEをキモトリプシンで限定加水分解すると、OYEは特異的に14K断片と、34K断片に切断され、この時に出現した14K断片のNー末端ロイシンのアミノ基が8ーフルオローFMMの8ー位を求核攻撃し、14K断片とフラビン部分が共有結合した。このことによって、OYEの一次構造上、および三次構造上の結合位置と結合様式が推定できた。

Research Products

(12 results)

All Other

All Publications (12 results)

[Publications] Retsu Miura: "19F-NMR study on the interaction of fluorobenzoate with pocine kidney D-amino acid oxidase." Journal of Biochemistry. 105. 318-322 (1989)
[Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by dicarboxylic acids." Biochemical and Biophysical Research Communications. 162. 168-174 (1989)
[Publications] 三浦洌: "フラビン酵素活性域への多核NMRの応用" 蛋白質核酸酵素. 35. 825-835 (1990)
[Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosohates." FEBS Letters. 264. 138-140 (1990)
[Publications] 藤井茂: "旧黄色酵素の活性域について" ビタミン. 64. 555-566 (1990)
[Publications] Shigeru Fujii: "Studies on the flavin-binding region of old yellow enzyme with an active site probe,8-fluoro-8-demethyl FMN." Journal of Biochemistry. 109. 55-60 (1991)
[Publications] Shigeru Fujiii: "On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P-and 13C-NMR.Use of 13C-enriched FAD as a probe." Journal of Biochemistry. 109. 144-149 (1991)
[Publications] Sabu Kasai: "Structure of FP390 including its prosthetic group(Q-flavin):physiological significance of light emitting reaction in luminous bacteria" Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 285-288 (1991)
[Publications] Kunio Matsui: "Photproduct of 8-hydroxyflavin." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 33-36 (1991)
[Publications] Shigeru Fujii: "13C-NMR study of NADPH-adrenodoxin reductase reconstituted with 13C-enriched FAD." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 381-384 (1991)
[Publications] Retsu Miura: "Stdies on old yellow enzyme reconstitutedwith the active site probe 8-fluoro-8-demethyl FMN.Covalent modification and 19F-NMR." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 357-360 (1991)
[Publications] Miho Ohta: "Bovine adrenal cytochrome P-450(11β)-mediated comversion of 11-Deoxycortisol to 18-and 19-hydroxy derivaties;structural analysis by 1H-NMR." Journal of Steroid Biochemistry. 39. 911-920 (1991)

1991 Fiscal Year Annual Research Report

多核NMR分光法によるフラビン酵素反応機構の研究

Principal Investigator

三浦 洌 関西医科大学, 医学部, 教授 (70093466)

Research Products

[Publications] Retsu Miura: "19F-NMR study on the interaction of fluorobenzoate with pocine kidney D-amino acid oxidase." Journal of Biochemistry. 105. 318-322 (1989)

[Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by dicarboxylic acids." Biochemical and Biophysical Research Communications. 162. 168-174 (1989)

[Publications] 三浦 洌: "フラビン酵素活性域への多核NMRの応用" 蛋白質核酸酵素. 35. 825-835 (1990)

[Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosohates." FEBS Letters. 264. 138-140 (1990)

[Publications] 藤井 茂: "旧黄色酵素の活性域について" ビタミン. 64. 555-566 (1990)

[Publications] Shigeru Fujii: "Studies on the flavin-binding region of old yellow enzyme with an active site probe,8-fluoro-8-demethyl FMN." Journal of Biochemistry. 109. 55-60 (1991)

[Publications] Shigeru Fujiii: "On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P-and 13C-NMR.Use of 13C-enriched FAD as a probe." Journal of Biochemistry. 109. 144-149 (1991)

[Publications] Sabu Kasai: "Structure of FP390 including its prosthetic group(Q-flavin):physiological significance of light emitting reaction in luminous bacteria" Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 285-288 (1991)

[Publications] Kunio Matsui: "Photproduct of 8-hydroxyflavin." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 33-36 (1991)

[Publications] Shigeru Fujii: "13C-NMR study of NADPH-adrenodoxin reductase reconstituted with 13C-enriched FAD." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 381-384 (1991)

[Publications] Retsu Miura: "Stdies on old yellow enzyme reconstitutedwith the active site probe 8-fluoro-8-demethyl FMN.Covalent modification and 19F-NMR." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 357-360 (1991)

[Publications] Miho Ohta: "Bovine adrenal cytochrome P-450(11β)-mediated comversion of 11-Deoxycortisol to 18-and 19-hydroxy derivaties;structural analysis by 1H-NMR." Journal of Steroid Biochemistry. 39. 911-920 (1991)

三浦洌関西医科大学, 医学部, 教授 (70093466)

[Publications] 三浦洌: "フラビン酵素活性域への多核NMRの応用" 蛋白質核酸酵素. 35. 825-835 (1990)

[Publications] 藤井茂: "旧黄色酵素の活性域について" ビタミン. 64. 555-566 (1990)