Catalysis by Prostaglandin Endoperoxide H Synthases

前列腺素内过氧化物 H 合成酶的催化作用

• 批准号: 7109363
• 负责人: William L Smith
• 金额: $ 43.77万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7109363
• 关键词: X ray crystallography; active sites; aminoacid; arachidonate; calorimetry; catalyst; cell line; enzyme activity; enzyme mechanism; gene targeting; genetic manipulation; genetically modified animals; isozymes; laboratory mouse; molecular dynamics; peroxidases; phenotype; prostaglandin endoperoxide synthase; protein structure function

DESCRIPTION (provided by applicant): Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs-1 and -2) catalyze the committed step in prostaglandin (PG) synthesis and are targets of nonsteroidal anti-inflammatory drugs and "COX-2 inhibitors". Each PGHS isoform subserves different physiological functions, but it is not known why it is necessary to have two isoforms. There are instances where PGHS-1 and -2 are co-expressed in cells at similar levels and the same subcellular sites and yet PGHS-2 produces PGs while PGHS-1 does not. These observations would argue against PGHS-2 being an inducible substitute or supplement for PGHS-1. More likely, the unique biological functions of PGHSs-1 and -2 result, at least in part, from structural/catalytic differences between the proteins themselves (i.e. PGHS-2 can do something that PGHS-1 cannot do, and conversely). PGHS-2 has three unique features: (a) an ability to oxygenate arachidonic acid (AA) esters efficiently; (b) the capacity to oxygenate AA at low concentrations of AA and activating peroxides; and (c) an 18 amino acid cassette near its C-terminus. There are two major goals of our proposed research. The first is to characterize further the basic structural and enzymatic properties of the cyclooxygenase and peroxidase activities of PGHSs-1 and -2. In the case of the cyclooxygenase, we will test predictions for the functions of specific active site amino acids in catalysis using a combination of mutagenic and x-ray crystallographic studies. With the peroxidase, we will use mutagenic, kinetic and computational studies to determine the basis for the unusual peroxide substrate specificity of PGHSs and for the difference between peroxide activation of the cyclooxygenase activities of PGHS-1 vs -2; in parallel, we will determine the roles of the peroxidase activities of PGHS-1 vs -2 in regulating cyclooxygenase activity in intact cells. Our second major goal is to determine if two of the distinctive properties of PGHS-2 (i.e. reactivity with AA esters or the C-terminal cassette) are essential for its novel biological actions. We will prepare and characterize the reproductive and developmental phenotypes of knock-in mice with mutations in their PGHS-2 gene such that they (a) can use AA but not esterified AA as a substrate (R120Q PGHS-2) or (b) lack the C-terminal cassette (delta581-598 PGHS-2) to determine if the knock-in mice are the same as PGHS-2 knock out mice.

描述（由申请人提供）：前列腺素内过氧化物H合酶-1和-2（PGHSS-1和-2）催化了前列腺素（PG）合成的一步，是非甾体类抗炎药的靶标，是“ COX-2抑制剂”的靶标。每个PGHs同工型都会产生不同的生理功能，但尚不知道为什么有两个同工型有必要。在某些情况下，PGHS-1和-2在相似水平和相同亚细胞位点的细胞中共表达，而PGHS-2则产生PGS，而PGHS-1则没有。这些观察结果将反对PGHS-2是PGHS-1的诱导替代或补充。更有可能，PGHSS-1和-2的独特生物学功能至少部分是由于蛋白质本身之间的结构/催化差异（即PGHS-2可以做PGHS-1无法做的事情，而是相反）。 PGHS-2具有三个独特的特征：（a）有效氧合蛛网膜酸（AA）酯有效的能力； （b）在低浓度的AA并激活过氧化物的能力； （c）其C末端附近的18个氨基酸盒。我们拟议的研究有两个主要目标。首先是进一步表征PGHSS -1和-2的环氧酶和过氧化物酶活性的基本结构和酶促特性。在环氧合酶的情况下，我们将使用诱变和X射线晶体学研究的组合来测试特定活性位点氨基酸在催化中的功能的预测。使用过氧化物酶，我们将使用诱变，动力学和计算研究来确定PGHSS的异常过氧化物底物特异性的基础，以及PGHS -1与-2的环氧酶活性的过氧化物活化之间的差异；同时，我们将确定PGHS -1 vs -2的过氧化物酶活性在调节完整细胞中环氧酶活性中的作用。我们的第二个主要目标是确定PGHS-2（即与AA酯或C末端盒的反应性）的两种独特性能对于其新型生物学作用至关重要。我们将准备并表征其PGHS-2基因中突变的敲击小鼠的生殖和发育表型，以便它们（a）可以使用AA，但不能使用AA作为底物（R120Q PGHS-2）或（B）缺乏C-terminal Cassette（Delta581-598 PGHS-PGHS-PGHS），以确定aggne of the Mongning Migne if periace if the per，如果cassette cassette cassette缺乏cassettal cassette cassette。