METALLOENZYME DYNAMICS AND MECHANISM

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。 这包括识别 这些血红素酶催化途径的基本步骤和 产生的催化中间体的检测和稳定 每个基本步骤。 这些目标很难实现，因为很多 基本步骤无法通过现有技术进行研究 正常情况。 在较低的温度下存在更多的催化中间体 短时间内的浓度。 为了克服这些问题， 停流冷冻酶学被用来检测和稳定新的 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，相互转化 这些酶的等电子、高价形式之间 用过氧化氢对铁酶进行初始氧化及其 随后的底物减少正在研究中。 微观率 将测量每个基本步骤的常数及其温度 用于表征热力学激活参数的依赖性 每个步骤以及每个步骤的相对热力学稳定性 中间。 基本速率常数值的变化 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 为蛋白质的这些部分赋予催化作用的目标。 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 和正铁肌红蛋白，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 将用于提供有关长寿结构的信息 中间体。 将为每种酶配制一种特定的化学物质 与观察到的催化途径和途径一致 氢过氧化物酶的功能。

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.