STRUCTURE AND MECHANISM OF PROSTAGLANDIN H2 SYNTHASE

描述：(改编自申请者摘要)本项目的目标 是发展对前列腺素H_2合酶 (PGHS)的功能集中在酶对抑制剂的识别和 脂肪酸底物及其与酶的关系 不同的活动。PGHS催化花生四烯酸转化为 前列腺素H_2和这一过程被非甾体激素抑制 抗炎药(NSAID)，如阿司匹林、布洛芬、萘普生和 消炎痛。该项目的具体目标是：(1)探索 活性中心可塑性：与构象络合的PGHS的结构 受限制的吲哚美辛类似物将被确定为研究 识别活性中心采用交替构象的能力 各种非甾体抗炎药，并更好地了解 环氧合酶和过氧化物酶活性部位相互联系。(2) 脂肪酸结合和环氧合酶催化作用的分析。的性质 花生四烯酸或相关脂肪酸的米氏络合物 将使用浸泡在衬底溶液中的晶体来测定PGHS 厌氧条件。结构数据将被用来阐明 底物与自由基酪氨酸385的空间关系 除了由酶施加的任何构象限制之外 底物。这一特定目标的目的是检查 脂肪酸底物和抑制剂与PGHS结合以测试Tyr-385 恰如其分地放置在充当氢原子抽取器的位置。特定的 目的(3)探讨过氧化物酶与酶活性的关系。 环氧合酶催化活性：结构将确定 过氧化物酶缺乏形式的PGHS(使用化学修饰技术 和/或与非铁金属卟啉重组。它们之间的比较 具有过氧化物酶活性形式的结构将 通过揭示有助于理解过氧化物酶的机制 底物结合/催化所需的活性部位的特征。 最后，不同环氧合酶或过氧化物酶的结构缺失 将确定酶的形式，以帮助澄清两者之间的关系 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.