STRUCTURE AND MECHANISM OF PROSTAGLANDIN H2 SYNTHASE

描述：（改编自申请人的摘要）本项目的目标 是为了了解前列腺素H2合酶 （PGHS）的功能集中在酶对抑制剂的识别， 脂肪酸底物和酶的两个之间的关系 不同的活动。 PGHS催化花生四烯酸转化为 前列腺素H2，这一过程受到非甾体类药物的抑制 抗炎药（NSAID），如阿司匹林、布洛芬、萘普生，和 吲哚美辛 该项目的具体目标是：（1）探索 活性部位可塑性：PGHS与构象复合的结构 将确定受约束的吲哚美辛类似物，以研究 活性位点在识别中采用交替构象的能力 各种NSAID，并更好地了解可能的机制， 环氧合酶和过氧化物酶活性位点相互通信。 （二） 脂肪酸结合和环氧合酶催化的分析。 的性质 花生四烯酸或相关脂肪酸的米氏络合物结合到 PGHS将使用浸泡在底物溶液中的晶体在 厌氧条件 结构数据将用于阐明 底物与自由基酪氨酸385的空间关系 除了由酶施加于蛋白质上的任何构象限制之外， 衬底 这一具体目标的目的是审查的结构， 与PGHS结合的脂肪酸底物和抑制剂，以测试tyr-385是否 位于合适的位置作为氢原子提取器。 具体 目的（3）探讨过氧化物酶与细胞凋亡的关系。 环氧合酶催化活性：结构将被确定 过氧化物酶缺乏型PGHS（使用化学修饰技术 和/或用非铁金属卟啉重构。 比较这些 酶的过氧化物酶感受态形式的结构将 有助于了解过氧化物酶的机制，揭示 底物结合/催化所必需的活性位点的特征。 最后，不同的环氧合酶或过氧化物酶的结构缺陷， 酶的形式将被确定，以帮助澄清关系 在这两个活动之间。

DESCRIPTION: (adapted from applicant's abstract) The goal of this project is to develop a structural understanding of how prostaglandin H2synthase (PGHS) functions focusing on both the enzyme's recognition of inhibitors and fatty acid substrates and on the relationship between the enzyme's two distinct activities. PGHS catalyzes the conversion of arachidonic acid to prostaglandin H2 and this process is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, naproxen, and indomethacin. The specific aims of the project are: (1) exploration of active site plasticity: structures of PGHS complexed with conformationally constrained indomethacin analogs will be determined to investigate the ability of the active site to adopt alternate conformations in recognizing various NSAIDs, and to better understand the possible mechanism by which the cyclooxygenase and peroxidase active sites communicate with each other. (2) Analysis of fatty acid binding and cyclooxygenase catalysis. The nature of the Michaelis complex of arachidonic acid or related fatty acids bound to PGHS will be determined using crystals soaked in substrate solutions under anaerobic conditions. The structural data will be used to elucidate the spatial relationship of the substrate with the radical tryosine 385 in addition to any conformational constraints imposed by the enzyme on the substrate. The purpose of this specific aim is to examine the structures of fatty acids substrates and inhibitors bound to PGHS to test whether tyr-385 is situated appropriately to act as a hydrogen atom abstractor. Specific Aim (3) Examination of the relationship between the peroxidase and cyclooxygenase catalytic activities: structures will be determined of peroxidase deficient forms of PGHS (using chemical modification techniques and/or reconstitution with non-iron metalloporphyrins. Comparison of these structures with that of the peroxidase competent form of the enzyme will contribute to the understanding of the peroxidase mechanism by revealing features of the active site necessary for substrate binding/catalysis. Finally, structures of different cyclooxygenase or peroxidase deficient forms of the enzyme will be determined to help clarify the relationship between the two activities.