CYCLOOXYGENASE CATALYSIS AND SUICIDE INACTIVATION

我们研究的目的是了解环氧合酶的作用机制。 PGH合成酶(PGHS)的催化作用。PGHS-1和-2催化形成 花生四烯酸中前列腺素过氧化氢(PGH/2)的研究 致力于前列腺素生物合成的一步。这两种酶都催化(A)a 环氧合酶反应，在该反应中，花生四烯酸转化为PGH/2和 (B)一种过氧化物酶反应，其中PGH/2被还原为PGH/2。 反应发生在不同但相互关联的环氧合酶和 被一组血红素一分为二的过氧化物酶。环氧合酶 活性中心是从膜上伸出的疏水通道 将PGHS的表面结合到酶的主要球状结构域。 脂肪酸底物和可能的02从膜进入这个位置 隧道的底部。过氧化物酶活性位于相反方向 蛋白质的表面，与髓过氧化物酶相似。我们的第一次 具体目的是表征氨基酸在人体内的作用 环氧合酶活性部位与花生四烯酸的结合。我们会 环氧合酶失活Y371F人的晶体结构测定 (H)活性部位结合花生四烯酸的PGHS-2突变体。我们还将 制备各种活性部位氨基酸的突变，并分析 这些变化对底物结合和催化的影响。我们的第二个 具体目的是确定环氧合酶的自杀失活 活性源于自由基引发的分子内蛋白质交叉 连接涉及Tyr504基团。我们将对多肽产品进行表征 从天然和自杀灭活的绵羊PGHS-1中提取 残基在失活过程中被修改。我们还将确定 形成的各种光谱中间体的形成速率 过氧化氢与H386A绵羊PGHS-1突变体PGHS-1的相互作用 没有经历失活。最后，我们将确定 取代了Tyr504。我们的第三个具体目标是确定H20 连接环氧合酶活性部位和细胞外基质的PGHS通道 蛋白质的外部充当质子的管道，即 从环氧合酶催化活性部位Tyr385中提取； 将表征可能阻断H20通道的突变(G227A、G536A) 以及预计会否定质子转移的突变(R37G，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).