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（由申请方提供）：前列腺素内过氧化物酶-1和-2（PGHS-1和-2）催化前列腺素（PG）合成中的关键步骤，是非甾体抗炎药和“考克斯-2抑制剂”的靶标。每种PGHS亚型都有不同的生理功能，但不知道为什么需要两种亚型。存在PGHS-1和PGHS-2在细胞中以相似的水平和相同的亚细胞位点共表达的情况，然而PGHS-2产生PG而PGHS-1不产生。这些观察结果将反对PGHS-2作为PGHS-1的可诱导替代物或补充物。更有可能的是，PGHS-1和PGHS-2的独特生物学功能至少部分是由蛋白质本身之间的结构/催化差异引起的（即PGHS-2可以做PGHS-1不能做的事情，反之亦然）。PGHS-2具有三个独特的特征：（a）有效地使花生四烯酸（AA）酯水解的能力;（B）在低浓度AA下使AA水解并活化过氧化物的能力;和（c）在其C-末端附近的18个氨基酸盒。我们提出的研究有两个主要目标。首先是进一步表征PGHS-1和PGHS-2的环氧合酶和过氧化物酶活性的基本结构和酶性质。在环加氧酶的情况下，我们将测试预测的功能的特定活性位点的氨基酸在催化使用组合的诱变和X-射线晶体学研究。对于过氧化物酶，我们将使用致突变、动力学和计算研究来确定PGHS异常过氧化物底物特异性的基础以及PGHS-1与-2的环氧合酶活性的过氧化物激活之间的差异;同时，我们将确定PGHS-1与-2的过氧化物酶活性在调节完整细胞中环氧合酶活性方面的作用。我们的第二个主要目标是确定PGHS-2的两个独特性质（即与AA酯或C-末端盒的反应性）是否对其新的生物学作用至关重要。我们将制备并表征PGHS-2基因突变的敲入小鼠的生殖和发育表型，以确定敲入小鼠是否与PGHS-2敲除小鼠相同，这些小鼠（a）可以使用AA但不能使用酯化AA作为底物（R120 Q PGHS-2）或（B）缺乏C末端盒（δ 581 -598 PGHS-2）。