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（申请人提供）：前列腺素内过氧化物 H 合酶-1 和 -2 (PGHS-1 和 -2) 催化前列腺素 (PG) 合成的关键步骤，是非甾体抗炎药和“COX-2 抑制剂”的靶标。每种 PGHS 同工型都具有不同的生理功能，但尚不清楚为什么需要有两种同工型。在某些情况下，PGHS-1 和 -2 在细胞中以相似的水平和相同的亚细胞位点共表达，但 PGHS-2 产生 PG，而 PGHS-1 则不产生。这些观察结果反对 PGHS-2 作为 PGHS-1 的诱导替代品或补充品。更有可能的是，PGHS-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/催化差异造成的（即PGHS-2可以做PGHS-1不能做的事情，反之亦然）。 PGHS-2 具有三个独特的功能：（a）能够有效氧化花生四烯酸（AA）酯； (b) 在低浓度 AA 下氧化 AA 并活化过氧化物的能力； (c) 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 PGHSs-1 和 -2 的环氧合酶和过氧化物酶活性的基本结构和酶学特性。就环氧合酶而言，我们将结合诱变和 X 射线晶体学研究来测试对催化中特定活性位点氨基酸功能的预测。对于过氧化物酶，我们将使用诱变、动力学和计算研究来确定 PGHS 不寻常的过氧化物底物特异性的基础，以及过氧化物激活 PGHS-1 与 -2 环氧合酶活性之间的差异；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中的作用。我们的第二个主要目标是确定 PGHS-2 的两个独特特性（即与 AA 酯或 C 末端盒的反应性）是否对其新颖的生物作用至关重要。我们将准备并表征 PGHS-2 基因突变的敲入小鼠的生殖和发育表型，以便它们 (a) 可以使用 AA 但不能使用酯化 AA 作为底物 (R120Q PGHS-2) 或 (b) 缺乏C 端盒 (delta581-598 PGHS-2) 以确定敲入小鼠是否与 PGHS-2 敲除小鼠相同。