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（PGHS）的作用机制。 工作模型是一个 自由基支链机理，其中酪氨酸自由基， 在血红素合酶与氢过氧化物相互作用后产生， 过氧化物酶催化循环，在生物合成中起中心催化作用。 环氧合酶反应的传播。 为了测试这种机制，我们 提议： 1.探讨了血红素的结构与功能的关系 辅基和载脂蛋白。 血红素结合机制将 研究使用几种光谱方法伴随酶 活动分析 一系列改性卟啉和卟啉 与铁以外的金属离子配位的金属离子将用作探针， 与载脂蛋白反应。 血红素结合化学计量 将首先通过血红素滴定的水平比较来评估 (or结合）使用由不同方法制备的载脂蛋白PGHS样品， 其次是纵向比较所使用的方法差异， 测量血红素的化学计量。2.详细描述了 每种酶活性，即，环氧合酶和过氧化物酶。 我们建议 使用瞬态动力学测量来获得重要的相关数据， 可通过光学、EPR或同位素监测的反应中间体 追踪器 花生四烯酸，环氧合酶的底物，以及一系列 过氧化物，包括过氧化氢、乙基过氧化氢、PCTG 2和 过氧化氢-二十碳四烯酸的各种位置异构体将 系统地检查。3.确定功能关键氨基酸 血红素蛋白中的残基 使用人cDNA克隆和定点 诱变，我们将专门测试突变的影响， 一对组氨酸和一个丝氨酸残基 活动 酪氨酸残基被认为是蛋白质之间的接头。 两种酶的活性首先是由过氧化物酶的活性产生的 然后作为环加氧酶的激活剂， 通过EPR分析，推测为血红素铁的轴向配体 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血红素的争议 化学计量，并获得有关相互作用细节的知识 血红素和脱辅基蛋白之间的联系瞬态动力学研究之二 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的时间事件的冲突。 第三种也是最后一种定点诱变方法将有助于 精确定位重要的氨基酸残基， 阐明了整个酶促反应机理。

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.