Xiao G, Tsai A L, Palmer G, Boyar W C, Marshall P J, Kulmacz R J
Department of Internal Medicine, University of Texas Health Science Center at Houston 77030, USA.
Biochemistry. 1997 Feb 18;36(7):1836-45. doi: 10.1021/bi962476u.
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.
有人提出，氢过氧化物诱导产生的酪氨酸自由基是前列腺素H合酶（PGHS-1）“基础”同工型的关键环氧化酶中间体。在本研究中，对于“诱导性”同工型（PGHS-2），也发现氢过氧化物能高产率地产生一种自由基，在g = 2.0058处有一个宽的单重峰（峰谷间距为29 G）。PGHS-2与酪氨酸修饰剂四硝基甲烷（TNM）反应，导致环氧化酶失活，并产生一个窄得多的自由基电子顺磁共振信号（峰谷间距为22 G）。添加环氧化酶抑制剂尼美舒利同样会导致PGHS-2自由基变窄。在PGHS-1中，用TNM进行酪氨酸硝化或用活性位点配体抑制环氧化酶会导致产生窄的电子顺磁共振信号而非宽的信号，这两种信号均源自真实的酪氨酸自由基，表明PGHS-2中氢过氧化物诱导产生的自由基也是酪氨酸自由基。先前已表明，用阿司匹林（乙酰水杨酸，ASA）处理PGHS-2会导致特定丝氨酸残基乙酰化、环氧化酶抑制以及脂氧合酶活性增加。相比之下，ASA使PGHS-1乙酰化会同时抑制脂氧合酶和环氧化酶活性。我们现在发现，经ASA处理的PGHS-2自由基与对照PGHS-2中的自由基无法区分。向经ASA处理的PGHS-2中添加尼美舒利会抑制脂氧合酶，并产生一个窄的自由基电子顺磁共振信号，类似于单独用TNM或尼美舒利处理的PGHS-2中所见的信号。因此，PGHS-2加氧酶活性的保留与天然自由基的保留相关，而活性的丧失与自由基的改变相关。天然的和经ASA处理的PGHS-2都只产生11-和15-HETE的R立体异构体，这表明脂氧合酶的立体化学并未因ASA而改变。天然的和经ASA处理的PGHS-2的脂氧合酶K(m)值远高于对照PGHS-2环氧化酶的K(m)值。综上所述，这些结果表明，相同的PGHS-2酪氨酸自由基作为环氧化酶和脂氧合酶催化的氧化剂，并且ASA使PGHS-2乙酰化有利于花生四烯酸以改变后的构象结合，这导致从C13提取前R氢并形成11(R)-和15(R)-HETE。
