PROSTAGLANDIN H SYNTHASE--REACTION MECHANISM

总体目标是提供对酶的分子理解。 前列腺素H合成酶的作用机制。工作模式是 自由基支链机制，其中酪氨酸自由基， 合成酶血红素与过氧化氢相互作用后产生的 过氧化物酶催化循环，在 环氧合酶反应的繁殖。为了测试这一机制，我们 建议： 1.研究血红素之间的结构-功能关系 修复体和脱脂蛋白。血红素的结合机制将是 几种光谱学方法与酶的结合研究 活动分析。一系列修饰的卟啉和卟啉 与铁以外的金属离子配位将用作探针 与本研究的载脂蛋白反应。血红素结合化学计量学 将通过首先对血红素滴定进行横向比较来评估 (或装订)使用从不同程序制备的载脂蛋白PGHS样品， 然后是对方法差异的纵向比较 测量血红素的化学计量比。2.刻画其具体作用机理 每种酶的活性，即环氧合酶和过氧化物酶。我们建议 使用瞬时动力学测量获得重要的相关数据 可用光学、电子顺磁共振或同位素监测的反应中间体 追踪剂。花生四烯酸，环氧合酶底物，以及一系列 过氧物包括过氧化氢、乙基过氧化氢、PCTG2和 羟基过氧二十碳四烯酸的各种位置异构体将被 系统地进行了检查。3.确定功能关键的氨基酸 这种血红素蛋白中的残留物。利用人的基因克隆和定点定位 突变，我们将专门测试突变对一种 特定的酪氨酸、一对组氨酸和酶上的一个丝氨酸残基 活动。酪氨酸残基被认为是 由过氧化物酶活性首次产生的两种酶活性 然后作为环氧合酶的激活剂，组氨酸对 被认为是通过EPR的血红素铁的轴向配体 研究；丝氨酸被确定为阿司匹林的乙酰化位点。 第一种具体的方法有助于解决关于亚铁血红素的争议 化学计量学和获得关于相互作用的细节的知识 在血红素和脱脂蛋白之间。瞬变动力学研究之二 将揭示PGHS的详细反应序列，并将有助于解决 目前观察到的酶催化时间事件之间的冲突。 第三种也是最后一种定点突变方法将有助于 准确定位重要的氨基酸残基，将增强 阐明了酶反应的整体机理。

The overall goal is to provide a molecular understanding of enzymic mechanism of Prostaglandin H synthase (PGHS). The working model is a free radical branched chain mechanism in which a tyrosine radical, generated after interaction of the synthase heme with hydroperoxide in the peroxidase catalytic cycle, serves a central catalytic role in the propagation of the cyclooxygenase reaction. To test this mechanism, we propose to: 1. Investigate the structure-function relationship between the heme prosthetic group and the apoprotein. Heme-binding mechanism will be studied using several spectroscopic methods accompanied with enzyme activity analysis. A series of modified porphyrins and porphyrins coordinated with metal ions other than iron will be used as probes to react with the apoprotein for this study. Heme binding stoichiometry will be assesed by first a horizontal comparison of the heme titration (or binding) using apo PGHS samples prepared from different procedures, followed by a vertical comparison of methodological differences used to measure the heme stoichiometry. 2. Characterize the detailed mechanism of each enzyme activity, i.e., cyclooxygenase and peroxidase. We propose to use transient kinetic measurements to obtain correlated data of important reaction intermediates which can be monitored by optical, EPR or isotope tracer. Arachidonate, substrate for the cyclooxygensase, and a series of peroxides including hydrogen peroxide, ethyl hydroperoxide, PCTG2 and various positional isomers of hydroperoxy-eicosateraenoic acid will be examined systematically. 3. Identify the functionally critical amino acid residues in this hemeprotein. Using a human cDNA clone and site-directed mutagenesis, we will specifically test the influence of mutation of a specific tyrosine, a pair of histidine and a serine residue on the enzyme activity. The tyrosine residue was proposed to be the linker between the two enzyme activities which is first generated by the peroxidase activity and then acts as an activator for the cyclooxygenase, the' histidine pair is proposed to be the axial ligands for the heme iron through EPR studies; and the serine is identified as the acetylation site of aspirin. The first specific approach helps to solve the controversy of heme stoichiometry and to gain knowledge about the details of the interaction between heme and the apoprotein. the second study by transient kinetics will reveal the detail reaction sequence of PGHS and will help resolve the current conflict of the observed temporal events of enzyme catalysis. The third and final approach of site-directed mutagenesis will help to pinpoint the important amino acid residues and Will enhance the elucidation of the overall enzymic reaction mechanism.