Structure/Function and Reaction Mechanism of PGHS

描述（由申请人提供）：本项目的总体目标是阐明前列腺素H合酶（PGHS）的结构/功能关系和反应机制，该酶在血管生理学和病理生理学中起关键作用。PGHS具有两种不同的酶活性：将花生四烯酸（AA）转化为PGG 2的环氧合酶和将PGG 2还原为PGH 2的过氧化物酶。一个自由基支链的机制，涉及一个关键的酪氨酰基自由基和底物衍生的自由基将进行测试，以阐明PGHS催化的自活化性能。本申请包括三个具体目标。本论文的第一个目的是利用快速冷冻EPR、ENDOR等技术，用同位素标记AA，对AA衍生的α-和烯丙基自由基的结构和动力学进行表征。初级和次级KIE将用于H-提取和烯丙基自由基形成。 第二个目的是测试的假设，无论是酪氨酰基自由基迁移或/和酪氨酰基自由基苯环旋转起着决定性的作用，在耦合的过氧化物酶环氧合酶催化。我们将区分酪氨酰基自由基苯环旋转机制从自由基迁移最小化后一种机制，使用单一和多个酪氨酸突变体定位替代酪氨酸参与自由基迁移。酪氨酰自由基构象动力学与过氧化物酶和环氧合酶催化之间的相关动力学测量。EPR、ENDOR和HFEPR将检测不同构象的冷冻捕获的酪氨酰自由基。 为了实现最后一个目标，血红素相对于微粒体膜表面的取向将通过取向膜上的线性二色性和EPR来确定，以解决底物呈递、产物释放以及PGHS与类花生酸生物合成中下游酶之间的通信问题。第一个目标是鉴定额外的中间体，阐明AA衍生自由基的结构变化和环加氧酶催化的机制。第二个目标将解决酪氨酰基自由基动力学对PGHS催化的作用。最后一个目的将提供直接的数据PGHS的膜拓扑结构和机制，其底物介绍和产品释放。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate structure/function relationships and reaction mechanism of prostaglandin H synthase (PGHS), which plays key roles in vascular physiology and pathophysiology. PGHS has two distinct enzyme activities: a cyclooxygenase that converts arachidonic acid (AA) to PGG2, and a peroxidase that reduces PGG2 to PGH2. A free radical branched-chain mechanism involving a key tyrosyl radical and substrate-derived radicals will be tested to elucidate the self-activation properties of PGHS catalysis. Three specific aims are included in this application. We will characterize the structure and kinetics of the AA-derived pentadienyl and allyl radical using specifically isotope labeled AA by rapid-freeze EPR, ENDOR in the first aim. Primary and secondary KIE will be employed for the H-abstraction and allyl radical formation. The second aim is to test the hypothesis that either tyrosyl radical migration or/and the tyrosyl radical phenyl ring rotation plays a decisive role in coupling the peroxidase to cyclooxygenase catalysis. We will differentiate the tyrosyl radical phenyl ring rotation mechanism from radical migration by minimizing the latter mechanism using single and multiple tyrosine mutants to locate the alternative tyrosine that participate in radical migration. Correlative kinetic measurements will be done between tyrosyl radical conformation dynamics with peroxidase and cyclooxygenase catalysis. EPR, ENDOR, and HFEPR will examine freeze-trapped tyrosyl radical at different conformation. To achieve the last aim, heme orientation relative to the microsome membrane surface will be determined by linear dichroism and EPR on the oriented membrane to address the issue on substrate presentation, product release and the communication between PGHS and downstream enzymes in eicosanoid biosynthesis. The 1st Aim is to identify additional intermediate(s), to elucidate the structure variation of AA-derived radical and the mechanism of the cyclooxygenase catalysis. The 2nd Aim will address the role of tyrosyl radical dynamics on PGHS catalysis. The last aim will provide direct data on the membrane topology of PGHS and the mechanism of its substrate presentation and product release.