METALLOENZYME DYNAMICS AND MECHANISM

金属酶动力学和机制

基本信息

批准号：
3274688
负责人：
HAROLD E VAN WART
金额：
$ 5.25万
依托单位：
SYNTEX (USA), INC.-RESEARCH DIVISION
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 1994-11-30
项目状态：
已结题

项目摘要

The long range goal of this research is to elucidate the catalytic pathway and mechanism of action of the hydroperoxidases horseradish peroxidase, cytochrome c peroxidase and catalase. This includes the identification of the elementary steps in the catalytic pathways of these heme enzymes and the detection and stabilization of the catalytic intermediates produced by each elementary step. These goals are difficult to achieve because many elementary steps are not accessible for study by current techniques under normal conditions. More catalytic intermediates are present at low concentrations for brief periods of time. To overcome these problems, stopped-flow cryoenzymology is being used to detect and stabilize new catalytic intermediates in these reactions, such as the newly discovered horseradish peroxidase compound O. In particular, the interconversion between isoelectronic, high-valent forms of these enzymes during both the initial oxidation of the ferric enzymes with hydrogen peroxide and their subsequent reductions by substrates are under study. The microscopic rate constants for each elementary step will be measured and their temperature dependencies used to characterize the thermodynamic activation parameters for each step and the relative thermodynamic stabilities of each intermediate. Changes in the values of the rate constants for elementary steps with pH will be investigated in order to assess whether they are influenced by the state of protonation of distal and proximal residues with the goal of assigning a catalytic role to these parts of the protein. In certain cases, parallel experiments will be carried out in Nalpha- acetylated microperoxidase-8 and met-myoglobin, since these species can serve as "models" for these hydroperoxidases. Resonance Raman spectroscopy will be used to provide information about structures of the longer lived intermediates. A specific chemical will be formulated for each enzyme that is consistent with the observed catalytic pathway and pathway and the function of the hydroperoxidase.

这项研究的远距离目标是阐明催化途径和氢过氧化物酶辣根过氧化物酶的作用机理，细胞色素C过氧化物酶和过氧化氢酶。这包括识别这些血红素酶和检测和稳定由每个基本步骤。这些目标很难实现，因为许多目标基本步骤无法通过当前技术进行研究正常情况。在低处存在更多的催化中间体短时间的浓度。为了克服这些问题，停止流量的冷冻酶学用于检测和稳定新这些反应中的催化中间体，例如新发现的辣根过氧化物酶化合物O。特别是互转换在这两个过程中，这些酶的等电子高价值形式之间用过氧化氢及其的初始氧化铁酶随后的底物减少正在研究。显微镜率将测量每个基本步骤的常数用于表征热力学激活参数的依赖项对于每个步骤和每个步骤的相对热力学稳定性中间的。基本速率常数的值变化将研究pH的步骤，以评估它们是否是受远端和近端残基质子化状态的影响为蛋白质的这些部分分配催化作用的目的。在某些情况，将在Nalpha-中进行并行实验乙酰化的微孔过氧化物酶8和Met-Myoglobin，因为这些物种可以充当这些氢过氧化物酶的“模型”。共振拉曼光谱将用于提供有关寿命更长的结构的信息中间人。每个酶都将制定一种特定的化学物质与观察到的催化途径和途径以及氢过氧化物酶的功能。