STRUCTURE/FUNCTION AND REACTION MECHANISM OF PGHS

描述：（改编自申请人的摘要）总体目标是提供 从分子水平了解前列腺素H的酶反应机制 合成酶（PGHS）。PGHS的工作模型是自由基支链 在这种机制中，酪氨酸自由基，产生后的相互作用， 合酶血红素与过氧化物酶催化循环中的氢过氧化物， 在环加氧酶的起始和增殖中的中心催化作用 反应同样的酪氨酰自由基也作为关键的中间体， 启动PGHS的自我灭活。 为了验证这些机理模型，我们建议：1）进行详细的研究 PGHS环加氧酶催化循环的研究 花生四烯酸将采用快速冷冻/EPR和快速淬灭/HPLC方法 用于定义费率和确定主要结构 中间体和酶产物。描述机械差异 Fe-PGHS和用Mn（II）或Mn（III）重构的PGHS之间 原卟啉2)阐明自失活发生的机制， 过氧化物酶和环氧合酶催化。快速扫描停流将 用于确定速率和识别关键中间体。第三章 通过定点突变评价结构-功能关系。的 参与过氧化物酶催化、自失活和酶促反应的关键残基 将研究自由基中间体的稳定性。 第一项研究有助于获得有关个人步骤的知识， 环氧合酶催化和关键反应的速率。第二项研究将 提供有关PGHS催化最终调节的有用信息 活动最后一项研究将指出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 对控制和调节生理和 与异甘草素相关的病理生理学事件。

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.