Catalysis by Prostaglandin Endoperoxide H Synthases

描述（由申请人提供）：前列腺素内过氧化物H合成酶-1和-2 （PGHSs-1和-2）催化前列腺素（PG）合成的特定步骤，是非甾体抗炎药和“COX-2抑制剂”的靶标。每个PGHS亚型具有不同的生理功能，但尚不清楚为什么需要两个亚型。在某些情况下，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，但PGHS-2产生pg，而PGHS-1不产生pg。这些观察结果将反对PGHS-2作为PGHS-1的诱导替代品或补充。更有可能的是，PGHSs-1和pghss -2的独特生物学功能至少部分是由于蛋白质本身的结构/催化差异（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)能够有效地氧化花生四烯酸（AA）酯；(b)在低浓度AA和活化过氧化物条件下氧化AA的能力；(c)靠近其c端的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的环加氧酶和过氧化物酶活性的基本结构和酶学性质。在环加氧酶的情况下，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PGHS-1与-2的过氧化物活化环加氧酶活性之间的差异；同时，我们将确定PGHS-1和pghs -2过氧化物酶活性在完整细胞中调节环加氧酶活性的作用。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端盒的反应性）是否对其新的生物作用至关重要。我们将准备和表征PGHS-2基因突变的敲入小鼠的生殖和发育表型，这样它们(a)可以使用AA但不使用酯化AA作为底物（R120Q PGHS-2）或(b)缺乏c端盒（delta581-598 PGHS-2）来确定敲入小鼠是否与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.