SPECTROSCOPIC STUDIES OF ENZYME-SUBSTRATE COMPLEXES

酶-底物复合物的光谱研究

基本信息

批准号：
3288902
负责人：
GEORGE H REED
金额：
$ 17.25万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-01-01 至 1994-12-31
项目状态：
已结题

项目摘要

Enzymes involved in the synthesis, utilization, and interconversion of phosphorylated metabolites require specific activation by inorganic cations. Reactions that involve metal-nucleotide complexes as substrates or effectors are of central importance in a wide range of cellular functions from metabolism to signal transduction. The goals of this project are to understand the relationship between the structures of enzyme-metal-substrate complexes and the basis for chemical activation of these molecules in enzymic catalysis. The research uses the spectroscopic methods of electron paramagnetic resonance, nuclear magnetic resonance, infrared, and synchrotron -x-ray spectroscopy, together with x-ray diffraction techniques, to determine structures of enzyme metal-substrate or inhibitor complexes with pyruvate kinase, creatine kinase, 3-phosphoglycerate kinase, and enolase. The specific aims of the project include: identification of the way by which pyruvate kinase exploits its three metal cofactors in activation of the transferrable phospho group of its substrates; the involvement of the metal cofactors in the activation of the keto add substrate for pyruvate kinase; high resolution x-ray crystallographic studies of crystalline complexes of rabbit muscle pyruvate kinase; studies of the roles of the two divalent cations at the active site of enolase in binding and activation of the substrate and in binding of inhibitors that mimic the carbanion intermediate in this reaction; an investigation of the thio acid analogue of 3-phosphoglycerate in activation of domain closure in 3phosphoglycerate kinase; and studies of linkage isomerism in ligands bound to the metal center in creatine kinase. Information on the structures of enzyme-substrate complexes is essential for understanding the fundamental chemistry of enzymic catalysis and for rational design of enzyme-specific inhibitors that can be used as therapeutic agents, or as biochemical and physiological reagents to manipulate, selectively, reactions or pathways.

参与合成、利用和相互转化的酶磷酸化代谢物需要无机物的特异性激活阳离子。涉及金属-核苷酸复合物作为底物的反应或效应器在广泛的细胞中具有核心重要性从新陈代谢到信号转导的功能。本次活动的目标项目的目的是了解结构之间的关系酶-金属-底物复合物及其化学活化的基础这些分子在酶催化作用下。该研究利用光谱电子顺磁共振、核磁共振方法、红外、同步加速器-X 射线光谱以及 X 射线衍射技术，以确定酶金属底物的结构或与丙酮酸激酶、肌酸激酶的抑制剂复合物， 3-磷酸甘油酸激酶和烯醇化酶。项目的具体目标包括：鉴定丙酮酸激酶利用其作用的方式三种金属辅助因子激活可转移磷酸基团它的基材；金属辅助因子参与激活丙酮酸激酶的酮添加底物；高分辨率 X 射线兔肌肉丙酮酸结晶复合物的晶体学研究激酶；研究活性位点上两种二价阳离子的作用烯醇化酶在底物的结合和活化以及结合模拟该反应中碳负离子中间体的抑制剂；一个 3-磷酸甘油酸硫代酸类似物活化作用的研究 3磷酸甘油酸激酶中的结构域闭合；和联系的研究肌酸激酶中与金属中心结合的配体的异构现象。有关酶-底物复合物结构的信息至关重要了解酶催化的基本化学并合理设计酶特异性抑制剂，可用作治疗剂，或作为生化和生理试剂有选择地操纵反应或途径。