MECHANISM OF THE GROUP TRANSFER PROCESS

团体转移流程的机制

基本信息

批准号：
3268015
负责人：
WILLIAM J RAY
金额：
$ 22.14万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-12-01 至 1992-11-30
项目状态：
已结题

项目摘要

A continuation of research on the catalytic mechanism of the phosphoglucomutase reaction is proposed that includes X-ray diffraction studies on the crystalline enzyme, as well as NMR (1H, 13C, 31P, and 113Cd), kinetic, and isotopic rate studies on solutions of the enzyme. These studies will focus on the mechanism that the enzyme uses to produce its large, catalytic rate effect. An electron density map with a nominal resolution of 2.7 Angstrom unit will be improved by molecular replacement techniques; the current molecular model will be modified and refined. Phases from the model plus additional diffraction data will be used to construct difference density maps of enzyme- substrate analog complexes after completing an evaluation of several possible complexes of this types, partly via NMR techniques. Related studies will explore 1H/1H interactions between bound substrate and the enzyme in substrate analog complexes. An attempt also will be made to observe and evaluate 1H/113Cd interactions between the bound metal ion and bound substrate. 31P-NMR studies on the interactions between the Cd2+ complex of the dephospho-enzyme and substrate analogs such as straight-chain 1,4-diol bisphosphates will be continued to further define how G1c-1, 6-P2 is bound by this form of the enzyme so that either the 1- or the 6-phosphate group is in position for transfer to a single acceptor group on the enzyme. A series of kinetic studies will be conducted with butane-1,4-diol monophosphate, which acts as an inefficient substrate (phosphate acceptor) to isolate the effects of pH and identity of the activating metal ion on the rate-controlling step in this process: the bond making/breaking step. A determination of the (18 O) kinetic isotope effect on the reaction (in the reverse direction, where (18 O)-labelled butane-1,4-diol-P2 is used to phosphorylate the enzyme) also will be conducted. Studies on how cosolutes affect the crystal growth of phosphoglucomutase at high salt will be continued by comparing the effects of polyethylene glycol and detergents on crystal growth rate. Procedures for reducing the salt content of such crystals to aid in the binding of substrates and analogs will be explored. Studies of how conditions used for data collection affect the quality of X-ray diffraction data also will be conducted.

继续研究有关催化机制的研究提出了包括X射线的磷酸葡萄糖酶反应晶体酶以及NMR的衍射研究（1H， 13c，31p和113cd），动力学和同位素率研究酶的溶液。这些研究将集中于酶用于产生其大型催化的机制费率效应。具有标称分辨率的电子密度图 2.7 Angstrom单元将通过分子置换来提高技术；当前的分子模型将被修改，并精制。来自模型的阶段以及其他衍射数据将用于构建酶的差异密度图完成评估后的底物模拟复合物这种类型的几种可能的复合物，部分是通过NMR 技术。相关研究将探索1H/1H的相互作用在衬底和底物类似物中的酶之间复合物。还将尝试观察和评估结合金属离子与结合之间的1H/113CD相互作用基材。 31P-NMR研究有关 Dephospho-酶和底物类似物的CD2+复合物例如直链1,4-二醇双磷酸盐将继续进行进一步定义了G1C-1，6-P2如何由这种形式的绑定酶使得1-或6-磷酸组在转移到酶上单个受体组的位置。一个将使用丁烷-1,4-二醇进行一系列动力学研究单磷酸盐，充当效率低下的底物（磷酸盐受体）隔离pH和身份的影响在此过程中激活金属离子在速率控制步骤上：债券建立/破坏步骤。（18 O）的确定动力学同位素对反应的影响（在相反的方向上，其中（18 o） - 标记的丁烷-1,4-二醇-P2用于磷酸化还将进行酶）。研究宇宙的研究影响高盐下磷酸葡萄糖酶的晶体生长通过比较聚乙烯乙二醇和晶体生长速率的洗涤剂。减少的程序这种晶体的盐含量有助于底物的结合和类似物将被探讨。研究条件如何用于数据收集也会影响X射线衍射数据的质量将进行。