Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2
Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2
复制标题
DOI：
10.1021/bi962476u
复制
发表时间：
1997-02-18
期刊：
影响因子：
2.9
通讯作者：
Kulmacz, RJ
中科院分区：
文献类型：
--
作者：
Xiao, GS;Tsai, AL;Kulmacz, RJ
过氧化氢诱导的酪氨酰自由基已被认为是前列腺素H合酶（PGHS-1）“基础"亚型的关键环氧合酶中间体。在本研究中，用“诱导型”异构体（PGHS-2），还发现氢过氧化物以高产率产生自由基，g = 2.0058（峰谷比为29 G）的宽单峰。PGHS-2与酪氨酸修饰剂四硝基甲烷（TNM）反应，导致环氧合酶失活和更窄的自由基EPR信号（22 G峰至谷）。加入环氧合酶抑制剂尼美舒利，同样导致了一个狭窄的PGHS-2自由基。在PGHS-1中，通过TNM或活性位点配体的酪氨酸硝化来抑制环氧合酶导致产生窄EPR而不是宽EPR，这两种信号都源自真实的酪氨酰基自由基，表明PGHS-2中的过氧化氢诱导的自由基也是酪氨酰基自由基。先前显示用阿司匹林（乙酰水杨酸，阿萨）处理PGHS-2导致特定丝氨酸残基的乙酰化、环氧合酶抑制和脂氧合酶活性增加。与此相反，乙酰化PGHS-1的阿萨，抑制脂氧合酶和环氧合酶的活性。我们现在已经发现ASA处理的PGHS-2自由基与对照PGHS-2中的自由基无法区分。另外尼美舒利ASA处理的PGHS-2抑制脂氧合酶，并导致一个狭窄的自由基EPR中看到的TNM或尼美舒利单独治疗的PGHS-2。因此，PGHS-2加氧酶活性的保留与天然自由基的保留有关，活性的丧失与自由基的改变有关。天然的和ASA处理的PGHS-2都只产生11-和15-HETE的R立体异构体，表明阿萨没有改变脂氧合酶的立体化学。天然和ASA处理的PGHS-2的脂氧合酶Km值显著高于对照PGHS-2环氧合酶。综上所述，这些结果表明，相同的PGHS-2酪氨酰基自由基作为环氧合酶和脂氧合酶催化的氧化剂，并且阿萨对PGHS-2的乙酰化有利于花生四烯酸以改变的构象结合，这导致从C13提取pro-R氢并形成11（R）-和15（R）-HETE。
A hydroperoxide-induced tyrosyl radical has been proposed as a key cyclooxygenase intermediate for the ''basal'' isoform of prostaglandin H synthase (PGHS-1). In the present study with the ''inducible'' isoform (PGHS-2), hydroperoxide was also found to generate a radical in high yield, a wide singlet at g = 2.0058 (29 G peak to trough). Reaction of PGHS-2 with a tyrosine-modifying reagent, tetranitromethane (TNM), resulted in cyclooxygenase inactivation and a much narrower radical EPR signal (22 G peak to trough). Addition of a cyclooxygenase inhibitor, nimesulide, similarly resulted in a narrow PGHS-2 radical. In PGHS-1, cyclooxygenase inhibition by tyrosine nitration with TNM or by active site ligands leads to generation of a narrow EPR instead of a wide EPR, with both signals originating from authentic tyrosyl radicals, indicating that the hydroperoxide-induced radicals in PGHS-2 are also tyrosyl radicals. Treatment of PGHS-2 with aspirin (acetyl salicylic acid, ASA) was previously shown to result in acetylation of a specific serine residue, cyclooxygenase inhibition, and increased lipoxygenase activity. Acetylation of PGHS-1 by ASA, in contrast, inhibited both lipoxygenase and cyclooxygenase activity. We now have found the ASA-treated PGHS-2 radical to be indistinguishable from that in control PGHS-2. Addition of nimesulide to ASA-treated PGHS-2 inhibited the lipoxygenase and resulted in a narrow radical EPR like that seen in PGHS-2 treated with TNM or nimesulide alone. Retention of PGHS-2 oxygenase activity was thus associated with retention of the native radical, and loss of activity was associated with alteration of the radical. Both native and ASA-treated PGHS-2 produced only the R stereoisomer of 11- and 15-HETE, demonstrating that the Lipoxygenase stereochemistry was not changed by ASA. Native and ASA-treated PGHS-2 had lipoxygenase K-m values considerably higher than that of the control PGHS-2 cyclooxygenase. Taken together, these results suggest that the same PGHS-2 tyrosyl radical serves as the oxidant for both cyclooxygenase and lipoxygenase catalysis and that acetylation of PGHS-2 by ASA favors arachidonate binding in an altered conformation which results in abstraction of the pro-R hydrogen from C13 and formation of 11(R)- and 15(R)-HETE.