CYCLOOXYGENASE CATALYSIS AND SUICIDE INACTIVATION

我们的研究目标是了解环氧合酶的作用机制 通过PGH脱氢酶（PGHS）催化。PGHS-1和PGHS-2催化 前列腺素内过氧化物H2（PGH/2）从花生四烯酸- 前列腺素生物合成的关键步骤。两种酶都催化（a）a 环氧合酶反应，其中花生四烯酸转化为PGH/2， (b)一种过氧化物酶反应，其中PGH/2被还原为PGH/2。这两 反应发生在不同但相互关联的环氧合酶和 过氧化物酶位点被血红素基团一分为二。环氧合酶 活性位点是从膜突出的疏水通道 PGHS的结合表面进入酶的主要球状结构域。 脂肪酸底物和可能的O2从膜进入该位点， 隧道的底部。过氧化物酶位点位于 蛋白质的表面，类似于髓过氧化物酶。我们的第一 具体目标是表征氨基酸在 环氧合酶活性位点结合花生四烯酸。我们将 确定环加氧酶失活的Y371 F人的晶体结构 (h)花生四烯酸结合在活性位点的PGHS-2突变体。我们还将 制备各种活性位点氨基酸的突变，并分析 这些变化对底物结合和催化的影响。我们的第二 具体目的是确定环加氧酶的自杀失活是否 活性来自自由基引发的分子内蛋白质交叉 涉及Tyr 504基团的连接。我们将表征肽产品 来自天然和自杀失活的绵羊PGHS-1，以确定 残基在失活期间被修饰。我们还将确定 各种光谱中间体的形成速率， 氢过氧化物与H386 A绵羊PGHS-1（一种突变PGHS， 无法进行灭活。最后，我们将确定影响f 取代Tyr 504。我们的第三个具体目标是确定H2O是否 PGHS通道，连接环氧合酶活性位点与 蛋白质的外部充当质子的管道， 从环氧合酶催化过程中的活性位点Tyr 385中提取;我们 将表征可能阻断H20通道的突变（G227 A，G536 A） 和预期否定质子转移的突变（R37 G，L）。

The goal of our research is to understand the mechanism of cyclooxygenase catalysis by PGH synthases (PGHSs). PGHS-1 and -2 catalyze the formation of prostaglandin endoperoxide H2 (PGH/2) from arachidonic acid--the committed step in prostaglandin biosynthesis. Both enzymes catalyze (a) a cyclooxygenase reaction in which arachidonate is converted to PGH/2 and (b) a peroxidase reaction in which PGH/2 is reduced to PGH/2. These two reactions occur at distinct but interconnected cyclooxygenase and peroxidase sites that are bisected by a heme group. The cyclooxygenase active site is a hydrophobic tunnel that protrudes from the membrane binding surface of PGHS into the major globular domain of the enzyme. Fatty acid substrates and probably 02 enter this site from the membrane at the base of the tunnel. The peroxidase site is located on the opposite surface of the protein and resembles that of myeloperoxidase. Our first specific aim to characterize the contributions of amino acids within the cyclooxygenase active site to the binding of arachidonic acid. We will determine the crystal structure of a cyclooxygenase-inactive Y371F human (h) PGHS-2 mutant with arachidonate bound in the active site. We will also prepare mutations of various active site amino acids and analyze the effects of these changes on substrate binding and catalysis. Our second specific aim is to determine if suicide inactivation of cyclooxygenase activity results from radical-initiated intramolecular protein cross linking involving a Tyr504 radical. We will characterize peptide products derived from native and suicide-inactivated ovine PGHS-1 to determine what residues are modified during inactivation. We will also determine the rates of formation of various spectral intermediates formed upon interaction of hydroperoxides with H386A ovine PGHS-1, a mutant PGHS that fails to undergo inactivation. Finally, we will determine the effect f replacing Tyr504. Our third specific aim is to determine if the H20 channel of PGHS that connects the cyclooxygenase active site with the exterior of the protein serves as a conduit for the proton that is abstracted from the active site Tyr385 during cyclooxygenase catalysis; we will characterize mutations likely to block the H20 channel (G227A, G536A) and mutations expected to negate proton transfer (R37G,L).