MECHANISM OF THE GROUP TRANSFER PROCESS

团体转移流程的机制

基本信息

批准号：
3268016
负责人：
WILLIAM J RAY
金额：
$ 29.79万
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 1996-11-30
项目状态：
已结题

项目摘要

A continuation of research aimed at describing the catalytic mechanism of the phosphoglucomutase reaction at the atomic level is posed. X-ray diffraction studies of the crystalline enzyme and complexes thereof will provide primary data. Supporting data will be obtained from solution studies of the following: the interaction between the enzymic phosphate group and the active site metal ion; bonding making/breaking in the transition state; the importance of selected amino acid side chains that line the catalytic cleft. One of the first goals will be to improve the current molecular model of the enzyme by collecting and processing higher-resolution diffraction data. A recently identified procedure that produces an improved lattice during elimination of the salt used in crystal growth will be developed further and employed. Use of this procedure should allow formation of various complexes of the crystalline enzyme that could not be obtained otherwise because of the salt present in untreated crystals. (The physical basis for the apparent annealing that accompanies this treatment will be probed, as a side issue -- and possibly its generality, along with procedures for improving the crystal growth process.) A variety of substrate, substrate analog, and transition state-analog complexes, also involving alternative metal ions, will be studied in the crystal phase by utilizing electron density- difference maps. The objective will be to identify the basis of the >1010-fold substrate binding-induced rate effect and the >1010-fold Mg2+- induced activation previously observed. An evaluation of bonding within the vanadate group of the transition-state analog complex via Raman spectroscopy and the use of EPR spectroscopy to assess binding-induced changes in interaction between bound Mn2+ and the phosphate group of the enzyme, after labelling it with 17O, and NMR spectrometry with the 113Cd enzyme will aid in interpreting X-ray diffraction results. An assessment of the 18O-kinetic isotope effect in a reaction where transfer of the (- PO32-) group of the enzyme to a suitable acceptor is rate-limiting also will facilitate interpretation of models obtained from X-ray diffraction studies, including that of a bound transition-state analog. The identity of several surface residues within the unusually large active site cleft of phosphoglucomutase will be altered by site-directed mutagenesis, with initial emphasis on residues close to the catalytic site that seem too far away to be involved directly in catalysis. Kinetically interesting mutant proteins will be subjected to a thorough physical/chemical evaluation.

旨在描述催化机制的研究的延续提出了原子水平的磷酸葡萄糖酶反应的。 X射线晶体酶及其复合物的衍射研究将提供主要数据。支持数据将从解决方案中获得以下研究：酶磷酸盐之间的相互作用组和活性位点金属离子；键合/破坏过渡状态；选定的氨基酸侧链的重要性在催化裂缝上排成一列。第一个目标之一是改善通过收集和加工的酶的当前分子模型高分辨率衍射数据。最近确定的程序在消除使用的盐时产生改进的格子晶体生长将进一步发展并使用。使用此程序应允许形成晶体的各种复合物由于存在的盐而无法获得的酶在未处理的晶体中。（明显退火的物理基础将伴随这种治疗方法作为附带问题 - 以及可能是其一般性，以及改善晶体的程序生长过程。）多种底物，底物类似物和过渡状态 - 分析复合物，也涉及替代金属离子，通过利用电子密度 - 差异图。目的是确定 > 1010倍底物结合诱导的速率效应和> 1010倍Mg2+ - 先前观察到的诱导激活。对键合的评估通过拉曼的过渡状态模拟复合物的杂料组光谱和EPR光谱法评估结合诱导的结合MN2+与磷酸组之间的相互作用变化用17O标记并用113CD标记NMR光谱后，酶酶将有助于解释X射线衍射结果。评估在一个反应中的18o-运动同位素效应中的转移（ - 酶的PO32-）与合适的受体一起限制速率将有助于解释从X射线衍射获得的模型研究，包括结合过渡状态类似物的研究。身份异常活跃部位裂缝中的几个表面残基磷酸葡萄糖酶的含量将通过位置定向的诱变改变，并且最初强调靠近催化部位的残留物远处直接参与催化。动力学有趣突变蛋白将经过彻底的物理/化学物质评估。