Molecular Design of Metalloproteins

金属蛋白的分子设计

• 批准号: 11228208
• 负责人: WATANABE Yoshihito
• 金额: $ 39.42万
• 依托单位: Nagoya University (2002-2003)Okazaki National Research Institutes (1999-2001)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11228208/
• 关键词: myoglobin; hydroxylation; cytochrome P450; 不斉酸化; シッフ塩基; 人工金属酵素; カタラーゼ; ヘム酵素; 活性酸素; 金属錯体; 酸化; ヘム蛋白質; ペルオキシダーゼ; 電子移動

The aromatic hydroxylation is one of the hard oxidations to proceed and we have designed a myoglobin mutant which is able to oxidize aromatic ring on the basis of the following strategies 1)Introduction of the substrate, tryptophan, at the position of phenyl alanine 43.2)Replacement of histidine 64 by asparitic acid to allow rapid reaction of the mutant with hydrogen peroxide. At the same time, Valine 68 was also replaced by isoleucine to allow hydrogen peroxide readily accessing to the active site, The oxidative modification of the protein was monitored by ESI-TOF mass measurements. Upon the addition of 1 eq. of hydrogen peroxide, 16Da increase of the molecular weight was observed. Further addition of hydrogen peroxide afforded secondary product exhibiting 32Da increase of its mass. After the digestion of the protein, a fragment including W43 was purified and characterized by MALDI TOF mass and NMR. The modified residue was W43 having 6-hydroxide. This is the first example of aromatic hydroxylation by a stoichiometric amount of hydrogen peroxide. In addition, the secondary product was characterized as 2,6-dioxo-derivative of tryptophan 43.

芳香族羟基化是进行的硬氧化之一，我们设计了一种能够氧化芳香环的肌红蛋白突变体，其基于以下策略1）在苯丙氨酸的位置引入底物色氨酸43.2）替换通过天冬氨酸对组氨酸 64 进行修饰，以允许突变体与过氧化氢快速反应。同时，缬氨酸68也被异亮氨酸取代，使得过氧化氢能够容易地到达活性位点，通过ESI-TOF质量测量来监测蛋白质的氧化修饰。添加1当量后。过氧化氢后，观察到分子量增加了 16Da。进一步添加过氧化氢得到二次产物，其质量增加了32Da。蛋白质消化后，纯化包含 W43 的片段并通过 MALDI TOF 质量和 NMR 进行表征。改性残基是具有6-氢氧化物的W43。这是通过化学计量的过氧化氢进行芳族羟基化的第一个例子。此外，次级产物被鉴定为色氨酸 43 的 2,6-二氧代衍生物。

项目成果

S.Ogo, T.Abura, Y.Watanabe: "pH-Dependent Transfer Hydrogenation of ketones with HCOONa as a Hydrogen Donor Promoted by (η^6-C_6Me_6)Ru Complexes"Organometallics. 21. 2964-2969 (2002)

S.Ogo、T.Abura、Y.Watanabe：“(η^6-C_6Me_6)Ru 配合物促进以 HCOONa 作为氢供体的 pH 依赖性酮转移氢化”有机金属学。

S.Ozaki, M.P.Roach, T.Matsui, Y.Watanabe: "Investigations of the Roles of the Distal Heme Environment and the Proximal Heme Iron Ligand in Peroxide Activation by Heme Enzymes via Molecular Engineering of Myoglobin"Acc.Chem.Res.. 34. 818-825 (2001)

S.Ozaki、M.P.Roach、T.Matsui、Y.Watanabe：“通过肌红蛋白分子工程研究远端血红素环境和近端血红素铁配体在血红素酶过氧化物活化中的作用”Acc.Chem.Res..

Y.Watanabe et al.: "Regioselective Hydroxylation of the Xylyl Linker in a Diiron(III) Complex having a carboxylate-Rich Ligand with H2O2"J.Chem.Soc., Chem.Commun.. 1900-1901 (2003)

Y.Watanabe 等人：“具有富含羧酸盐的配体与 H2O2 的二铁（III）配合物中的二甲苯连接体与 H2O2 的区域选择性羟基化”J.Chem.Soc.，Chem.Commun.. 1900-1901 (2003)

Y.Watanabe et al.: "Mechanisms of Hydrogen-, Oxygen-, and Electron-Transfer Rea Lions of Cumylperoxy Radial"J.Am.Chem.Soc. 125. 9074-9082 (2003)

Y.Watanabe 等：“过氧化枯基径向氢、氧和电子转移反应的机制”J.Am.Chem.Soc。

H.Nakai, S.Ogo, Y.Watanabe: "pH-Dependent Cross-Coupling reactions of water-Soluble organic halides with organoboron Compounds Catalyzed by the organometallic Aqua Complex [(SCS)Pd^<II>(H_2O)]^+ (SCS=C_6H_3-2,6-(CH_2SBut)_2)"Organometallics. 21. 1674-1678

H.Nakai、S.Ogo、Y.Watanabe：“由有机金属水复合物 [(SCS)Pd^<II>(H_2O)]^ 催化的水溶性有机卤化物与有机硼化合物的 pH 依赖性交叉偶联反应