Research of the structural factors for eliciting the highly catalytic efficiency of cold-active enzyme

冷活性酶高催化效率的结构因素研究

• 批准号: 17613001
• 负责人: TSURUTA Hiroki
• 金额: $ 2.24万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17613001/
• 关键词: low temperature environment; cold-active enzyme; phosphatase; X-ray structural analysis; hydrophobic interaction; 低温活性機構; 変異型酵素解析; 高分解能X線結晶構造解析; 変異体ライブラリー

Our final goal is that the investigation of the structural factor(s) for eliciting the highly catalytic efficiency of cold-active enzymes. For the purpose, we focused on cold-active protein-tyorosine phopshtase (CAPTPase) of a psychrophile Shewanella sp. as a experimental material. CAPTPase is a novel protein tyrosine phosphatase with the phosphoesterase motif conserved among metallophosphoesterases including many protein serine/threonine phosphatases. In this study, firstly, in order to reveal the detailed structural features and the reaction mechanism of this enzyme, we specified the metal ions in the enzyme protein and determined the crystal structure of CAPTPase at resolution of 1.1 Å. As the results, it was obvious that CAPTPase had two Zn atoms (termed Zn1 and Zn2) constructing a dinuclear Zn-Zn center in the catalytic site and that the hydrolysis of this enzyme was initiated by the nucleophilic attack with a water molecule bridging two Zn atoms after the substrate binding. The loop structures in the dinuclear Zn-Zn center were lined mainly with hydrophobic interactions. The mutational analysis for these hydrophobic residues and the determined crystal structure of I115M mutant enzyme, of which the catalytic efficiency was higher than that of the wild type enzyme, indicated that the catalysis of CAPTPase was dependent on the chemical bond force of hydrophobic interactions behind the loop structures contributing to the configuration of Zn1 atom.

我们的最终目标是研究引起冷活性酶高催化效率的结构因素。为此，我们以嗜冷菌希瓦氏菌（Shewanella sp.）的冷活性蛋白-酪氨磷酸酶（CAPTPase）为实验材料。CAPTlase是一种新的蛋白酪氨酸磷酸酶，其磷酸酯酶基序在包括许多蛋白丝氨酸/苏氨酸磷酸酶的金属磷酸酯酶中是保守的。在本研究中，首先，为了揭示该酶的详细结构特征和反应机理，我们确定了酶蛋白中的金属离子，并在1.1 nm分辨率下测定了CAPTlastine的晶体结构。结果表明，CAPTlase的催化位点上有两个Zn原子（称为Zn 1和Zn 2），构成了一个双核Zn-Zn中心，该酶的水解反应是在底物结合后，由一个水分子桥接两个Zn原子进行亲核攻击而引发的.双核Zn-Zn中心的环状结构主要由疏水相互作用形成。对这些疏水残基的突变分析和I115 M突变酶的晶体结构测定表明，CAPTlase的催化作用依赖于环结构背后的疏水相互作用的化学键力，而环结构对Zn 1原子的构型有贡献。

项目成果

Enzymatic characteristics of cold-active alkaline phosphatase

冷活性碱性磷酸酶的酶学特性

• 发表时间: 2006
• 期刊: Memoirs of graduate school of science and technology, Kobe university 24-A
• 作者: Nunomura;S.;Kitanaka;S.;Ra;C.;小笠原正明(他5名);Haruko Yamaguchi
• 通讯作者: Haruko Yamaguchi