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43.2的位置引入底物色氨酸43.2）用天冬氨酸替换组氨酸64以使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

Y.Watanabe et al.: "Isolation and Crystal Structure of Water-Soluble Iridium Hydride. Robin and Highly Active Catalyst for Acid-Catalyzed Transfer Hydrogenation of Carbonyl Compounds in Acidic Media"J.Am.Chem.Soc. 125. 4149-4154 (2003)

Y.Watanabe 等人：“水溶性氢化铱的分离和晶体结构。罗宾和酸性介质中羰基化合物酸催化转移氢化的高活性催化剂”J.Am.Chem.Soc。

T.Ueno, M.Suzuki, T.Goto, T.Matsumoto, K.Nagayama, Y.Watanabe: "Size Selective Olefin Hydrogenation by a Pd Nanocluster Provided in the Apo-Ferritin Cage"Angew. Chem. Int. Ed.. 43. 2527-2530 (2004)

T.Ueno、M.Suzuki、T.Goto、T.Matsumoto、K.Nagayama、Y.Watanabe：“载脂铁蛋白笼中提供的 Pd 纳米团簇进行尺寸选择性烯烃氢化”Angew。

T.Abura, S.Ogo, Y.Watanabe, S.Fukuzumi: "Isolation and Crystal Structure of Water-Soluble Iridium Hydride. Robust and Highly Active Catalyst for Acid-Catalyzed Transfer Hydrogenations of Carbonyl Compounds in Acidic Media"J.Am.Chem.Soc.. 125(印刷中). (2003)

T.Abura、S.Ogo、Y.Watanabe、S.Fukuzumi：“水溶性氢化铱的分离和晶体结构。酸性介质中羰基化合物酸催化转移氢化的稳健且高活性的催化剂”J.Am. Chem.Soc.. 125（印刷中）（2003）。

S.Ozaki, I.Hara, T.Matsui, Y.Watanabe: "Molecular Engineering of myoglobin : The Improvement of Oxidation Activity by Replacing Phe-43 with Tryptophan"Biochemistry. 40. 1044-1052 (2001)

S.Ozaki、I.Hara、T.Matsui、Y.Watanabe：“肌红蛋白的分子工程：用色氨酸替换 Phe-43 来提高氧化活性”生物化学。

M.P.Roach,S.Ozaki,Y.Watanabe: "Investigations of the Myoglobin Cavity Mutant H93G with Unnatural Imidazole Proximal Ligands as a Modular Peroxide O-O Bond Cleavage Model System"Biochemistry. 39. 1446-1454 (2000)

M.P.Roach、S.Ozaki、Y.Watanabe：“用非天然咪唑近端配体作为模块化过氧化物 O-O 键裂解模型系统研究肌红蛋白腔突变体 H93G”生物化学。