METALLOENZYME DYNAMICS AND MECHANISM

金属酶动力学和机制

基本信息

批准号：
3274683
负责人：
HAROLD E VAN WART
金额：
$ 7.65万
依托单位：
FLORIDA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-01-01 至 1986-06-30
项目状态：
已结题

项目摘要

The dynamic interaction of an enzyme with its substrate occurs along a well-defined catalytic pathway that consists of multiple, elementary steps. As this pathway is traversed, transient enzyme-substrate (E . S) intermediates are formed. In order to fully understand the mechanism of enzyme catalysis, it is essential that the structures of both the enzyme and substrate in each of these intermediates be elucidated. In the past, studies of E . S intermediates have proven difficult because of their brief lifetime andlow concentration. Furthermore, general methods capable of providing information about structure in solution have not been developed. The proposed research combines two areas of current research to overcome these problems. Subzero temperatures and cryosolvents will be used to accumulate intermediates in high concentrations for periods of time that permit their spectroscopic study. The spectroscopy will then be used to study intermediates in which either the substrate or enzyme is chromophoric to yield vibrational spectra of parts of the active site. This research will focus on metalloenzymes because they often have chromophoric centers that are ideally suited for resonance Raman studies. In one series of experiments, the rR spectra of intermediates in the hydrolysis of chromophoric peptide and dithioester substrates by leucine amino-peptidase will be studied to detect structural changes in the substrate during catalysis. In other experiments, rR spectra of intermediates formed during the oxidation of colorless substrates by the chromophoric enzyme horse radish peroxidase will be examined to detect changes in the structure of the heme group as it participates in catalysis.

酶与其底物的动态相互作用沿A发生定义明确的催化途径由多个小学组成步骤。随着该途径的遍历，瞬态酶 - 底物（例如S）中间体形成。为了充分理解酶催化，至关重要的是两种酶的结构这些中间体中每个中间体中的底物得到阐明。在过去， e的研究。事实证明，S中间体很难生命周期和水的浓度。此外，一般方法提供有关解决方案结构的信息尚未开发。拟议的研究结合了当前研究的两个领域，以克服这些问题。亚零温度和冷冻溶剂将用于在高浓度的时间内积累中间体允许他们的光谱研究。光谱法将用于研究中间体，其中底物或酶是发色的产生活性位点部分的振动光谱。这项研究将专注于金属酶，因为它们经常具有发色化中心非常适合共鸣拉曼研究。一系列实验，中间体在水解中的RR光谱亮氨酸氨基肽酶的彩色肽和二硫蛋白底物将研究以检测底物的结构变化催化。在其他实验中，在此期间形成的中间体的RR光谱发色酶马对无色底物的氧化将检查萝卜过氧化物酶以检测结构的变化血红素小组参与催化。