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Studies on the reaction mechanism of peroxidase based on its tertiary structure

基于过氧化物酶三级结构的反应机理研究

基本信息

批准号：
04680055
负责人：
FUKUYAMA Keiichi
金额：
$ 1.09万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04680055/
关键词：
peroxidase X-ray crystallography three-dimensional structure

项目摘要

The reaction mechanism of peroxidase from a fungus Arthromyces ramosus (ARP) has been discussed on the basis of its three-dimensional structure determined by X-ray crystallography.1. The crystal structure of ARP has been determined by the multiple isomorphous replacement method and refined to R=17.4 % for the 19,191 reflections between 7.0 and 1.9 A resolution. The model includes residues 9 to 344, the heme group, two N-acetylglucosamine residues, two calcium ions and 246 water molecules.2. The overall tertiary structure of ARP is similar to that of yeast cytochrome c peroxidase (CCP) and that of the lignin peroxidase from Phanerochaete chrysosporium. Although the histidine and arginine residues (His56 and Arg52) at the distal side of the heme are conserved in the amino acid sequences of various heme peroxidases, the orientations of the side chains of these residues relative to the heme in ARP differ significantly from those in CCP and Lip.In order to explore the role of Arg52 and His56 during the compound I formation, the crystal structure of ARP complexed with a substrate analogue I_3 - has been determined at 2.2 A resolution. Upon I_3" binding the side chain of Arg52 moved little, while that of His56 moved significantly. On the basis of these results the mode of binding of peroxide to the heme has been similated with computer graphics, and the process of the enzyme reaction as well as the role of these residues has been discussed.

利用x射线晶体学对真菌长节真菌（Arthromyces ramosus， ARP）过氧化物酶的三维结构进行了分析，探讨了过氧化物酶的反应机理。用多重同构置换法确定了ARP的晶体结构，并对7.0 ~ 1.9 A分辨率的19191次反射进行了R= 17.4%的细化。该模型包括9 ~ 344残基、血红素基、两个n -乙酰氨基葡萄糖残基、两个钙离子和246个水分子。ARP的三级结构与酵母细胞色素c过氧化物酶（CCP）和黄孢原毛毛菌的木质素过氧化物酶相似。尽管在各种血红素过氧化物酶的氨基酸序列中，位于血红素远端的组氨酸和精氨酸残基（His56和Arg52）是保守的，但ARP中这些残基相对于血红素的侧链取向与CCP和Lip有显著不同。为了探索Arg52和His56在化合物I形成过程中的作用，在2.2 a分辨率下测定了ARP与底物类似物I_3 -络合的晶体结构。当I_3结合时，Arg52的侧链移动不大，而His56的侧链移动明显。在这些结果的基础上，用计算机图形模拟了过氧化物与血红素的结合方式，并讨论了酶反应的过程以及这些残基的作用。