Catalysis by Prostaglandin Endoperoxide H Synthases

描述(申请人提供)：前列腺素内源性过氧化氢合成酶-1和-2(PGHSs-1和-2)催化前列腺素(PG)合成的关键步骤，是非类固醇抗炎药和“COX-2抑制剂”的靶标。每一种PGHS亚型具有不同的生理功能，但目前尚不清楚为什么必须有两种亚型。有些情况下，PGHS-1和-2在相同水平和相同的亚细胞位置的细胞中共表达，但PGHS-2产生PGs，而PGHS-1不产生PGs。这些观察结果将反对PGHS-2作为PGHS-1的诱导性替代品或补充。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的结构/催化差异造成的(即PGHS-2可以做一些PGHS-1做不到的事情，反之亦然)。PGHS-2有三个独特的特征：(A)能够有效地氧化花生四烯酸(AA)酯；(B)能够在低浓度的AA和活化的过氧化物中氧化AA；以及(C)在其C末端附近有一个18个氨基酸的盒。我们提出的研究有两个主要目标。一是进一步鉴定PGHSs-1和PGHSs-2的环氧合酶和过氧化物酶活性的基本结构和酶性质。在环氧合酶的情况下，我们将使用诱变和X射线结晶学研究相结合的方法来测试特定活性部位氨基酸在催化中的功能预测。对于过氧化物酶，我们将利用诱变、动力学和计算研究来确定PGHS不寻常的过氧化物酶底物专一性的基础以及过氧化氢激活PGHS-1VS-2环氧合酶活性的差异；同时，我们将确定PGHS-1VS-2的过氧化物酶活性在调节完整细胞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的两个独特的性质(即与AA酯或C端盒的反应性)是否对其新的生物学作用是必要的。我们将制备和鉴定其PGHS-2基因突变的敲除小鼠的生殖和发育表型，以便(A)可以使用AA而不是酯化的AA作为底物(R120Q PGHS-2)或(B)缺乏C-末端盒(delta581-598PGHS-2)以确定敲除小鼠是否与PGHS-2敲除小鼠相同。

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.