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.

芳香族羟基化是难进行的氧化反应之一，我们基于以下策略设计了一种能够氧化芳香环的肌红蛋白突变体：1)在苯丙氨酸位置引入底物色氨酸；4)用天冬氨酸取代组氨酸，使突变体能够与过氧化氢快速反应。同时，Valine 68也被异亮氨酸取代，使过氧化氢容易进入活性部位，通过ESI-TOF质量测量监测了蛋白质的氧化修饰。在增加1等式后过氧化氢的相对分子质量增加了16Da。过氧化氢的进一步加入使二次产物的质量增加了32Da。经酶切后，纯化出含有W43的片段，并用MALDI、TOF、MS和核磁共振对其进行了表征。改性残留物为含6-羟基的W43。这是第一个通过化学计量的过氧化氢进行芳香族羟基化的例子。此外，二次产物经鉴定为色氨酸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。