METALLOENZYME DYNAMICS AND MECHANISM

金属酶动力学和机制

• 批准号: 2174913
• 负责人: HAROLD E VAN WART
• 金额: $ 13.3万
• 依托单位: SYNTEX (USA), INC.-RESEARCH DIVISION
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-01-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174913
• 关键词: Cruciferae; Raman spectrometry; catalase; catalyst; chemical structure function; chromophore; circular magnetic dichroism; conformation; cryoscience; cytochrome c; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; horseradish peroxidase; metalloenzyme; solutions; solvents; thermodynamics

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.

这项研究的长期目标是阐明催化途径 以及氢过氧化物酶辣根过氧化物酶的作用机制， 细胞色素C过氧化物酶和过氧化氢酶。 这包括确定 这些血红素酶的催化途径中的基本步骤， 检测和稳定催化中间体产生的 每一个基本步骤。 这些目标很难实现，因为许多 基本步骤不能通过现有技术进行研究 正常情况下。 更多的催化中间体存在于较低的 短时间内的浓度。 为了克服这些问题， 停流冷冻酶学正被用于检测和稳定新的 这些反应中的催化中间体，如新发现的 辣根过氧化物酶复合物O. 特别是， 这些酶的等电子，高价形式之间， 铁酶与过氧化氢的初始氧化及其 正在研究随后通过底物的还原。 是两种微观速率 将测量每个基本步骤的常数， 用于表征热力学活化参数的相关性 对于每个步骤和每个步骤的相对热力学稳定性， 中间体 在变化的速率常数的值为小学 将研究pH值的步骤，以评估它们是否 受远端和近端残基质子化状态的影响， 目的是赋予蛋白质的这些部分催化作用。 在 在某些情况下，将在Nalpha中进行平行实验- 乙酰化微过氧化物酶-8和高铁肌红蛋白，因为这些物种可以 作为这些氢过氧化物酶的“模型”。 共振拉曼光谱 将被用来提供有关结构的信息， 中间体的 将为每种酶配制一种特定的化学品， 与观察到的催化途径和途径一致， 氢过氧化物酶的功能。