STRUCTURE/FUNCTION AND REACTION MECHANISM OF PGHS

描述：(改编自申请者摘要)总体目标是提供 前列腺素H酶反应机理的分子理解 合酶(PGHS)。PGHS的工作模式是自由基支链 酪氨酸基的作用机制 在过氧化物酶催化循环中，合成酶血红素与过氧化氢一起服务于 环氧合酶的启动和增殖中的中心催化作用 反应。同样的酪氨酸基也是关键的中间体 启动PGHS的自我失活。 为了检验这些机理模型，我们建议：1)进行详细的研究 一系列特异性标记的PGHS环加氧酶催化循环的研究 花生四烯酸。快速冷冻/EPR和快速冷却/高效液相色谱方法将是 用来定义费率和确定主要的 中间体和酶产品。描述机理上的差异 Fe-PGHS和由Mn(II)或Mn(III)重组的PGHS之间的关系 原卟啉。2)阐明了自失活发生的机理。 过氧化物酶和环氧合酶都有催化作用。快速扫描停止流将 被用来确定价格和确定关键中间体。3) 通过定点突变评价结构与功能的关系。这个 关键残基参与过氧化物酶催化、自失活和 自由基中间体的稳定性将被考察。 第一项研究有助于获得关于以下各个步骤的知识 环氧合酶催化和关键反应速率。第二项研究将 提供有关PGHS催化最终调控的有用信息 活动。最后一项研究将确定蛋白质中的关键元素， 在催化和失活中发挥特定的功能作用。了解 PGHS在心血管系统中的基本反应机制应提供 对生理和心理状态的控制和调节有很深的了解 与前列腺素相关的病理生理事件。

DESCRIPTION: (adapted from applicant's abstract) The overall goal is to provide a molecular understanding the enzyme reaction mechanism of Prostaglandin H synthase (PGHS). The working model for PGHS 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 initiation and propagation of the cyclooxygenase reaction. This same tyrosyl radical also serves as the key intermediate to launch the self-inactivation of PGHS. To test these mechanistic models, we propose to: 1) Carry out detailed studies of the PGHS cyclooxygenase catalytic cycle using series of specific-labeled arachidonic acid. Rapid-freeze/EPR and rapid-quenching/HPLC methods will be employed to define the rates and to determine the structure of the main intermediates and enzyme products. Characterize the mechanistic differences between the Fe-PGHS and PGHS reconstituted with Mn(II) or Mn(III) protoporphyrins. 2) Elucidate the mechanism of self-inactivation occurring in both the peroxidase and cyclooxygenase catalysis. Rapid-scan stopped-flow will be used to determine the rates and to identify the key intermediates. 3) Evaluate the structure-function relationships by site-directed mutagenesis. The key residues involved in the peroxidase catalysis, self-inactivation and the stability of the radical intermediates will be investigated. The first study helps to gain knowledge about the individual steps of cyclooxygenase catalysis and the rates of key reactions. The second study will provide useful information about the ultimate regulation of PGHS catalytic activity. The last study will pin point the key elements in the protein that play specific functional roles in catalysis and inactivation. Understanding the basic reaction mechanism of PGHS in the cardiovascular systems should provide great insight into the control and regulation of the physiological and pathophysiological events associated with prostaglandins.