Tsai Ah-lim, Palmer Graham, Wu Gang, Peng Sheng, Okeley Nicole M, van der Donk Wilfred A, Kulmacz Richard J
Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
J Biol Chem. 2002 Oct 11;277(41):38311-21. doi: 10.1074/jbc.M206961200. Epub 2002 Aug 6.
Peroxide-generated tyrosyl radicals in both prostaglandin H synthase (PGHS) isozymes have been demonstrated to couple the peroxidase and cyclooxygenase activities by serving as the immediate oxidant for arachidonic acid (AA) in cyclooxygenase catalysis. Acetylation of Ser-530 of PGHS-1 by aspirin abolishes all oxygenase activity and transforms the peroxide-induced tyrosyl radical from a functional 33-35-gauss (G) wide doublet/wide singlet to a 26-G narrow singlet unable to oxidize AA. In contrast, aspirin-treated PGHS-2 (ASA-PGHS-2) no longer forms prostaglandins but retains oxygenase activity forming 11(R)- and 15(R)-hydroperoxyeicosatetraenoic acid and also retains the EPR line-shape of the native peroxide-induced 29-30-G wide singlet radical. To evaluate the functional role of the wide singlet radical in ASA-PGHS-2, we have examined the ability of this radical to oxidize AA in single-turnover EPR studies. Anaerobic addition of AA to ASA-PGHS-2 immediately after formation of the wide singlet radical generated either a 7-line EPR signal similar to the pentadienyl AA radical obtained in native PGHS-2 or a 26-28-G singlet radical. These EPR signals could be accounted for by a pentadienyl radical and a strained allyl radical, respectively. Experiments using 11d-AA, 13(R)d-AA, 15d-AA, 13,15d(2)-AA, and octadeuterated AA (d(8)-AA) confirmed that the unpaired electron in the pentadienyl radical is delocalized over C11, C13, and C15. A 6-line EPR radical was observed when 16d(2)-AA was used, indicating only one strongly interacting C16 hydrogen. These results support a functional role for peroxide-generated tyrosyl radicals in lipoxygenase catalysis by ASA-PGHS-2 and also indicate that the AA radical in ASA-PGHS-2 is more constrained than the corresponding radical in native PGHS-2.
已证明，在两种前列腺素H合酶（PGHS）同工酶中，过氧化物生成的酪氨酰自由基通过作为环氧化酶催化中花生四烯酸（AA）的直接氧化剂，将过氧化物酶和环氧化酶活性偶联起来。阿司匹林对PGHS-1的Ser-530进行乙酰化会消除所有加氧酶活性，并将过氧化物诱导的酪氨酰自由基从功能性的33 - 35高斯（G）宽双峰/宽单峰转变为无法氧化AA的26 - G窄单峰。相比之下，经阿司匹林处理的PGHS-2（ASA-PGHS-2）不再形成前列腺素，但保留加氧酶活性，生成11(R)-和15(R)-氢过氧化二十碳四烯酸，并且还保留了天然过氧化物诱导的29 - 30 - G宽单峰自由基的电子顺磁共振（EPR）线形。为了评估宽单峰自由基在ASA-PGHS-2中的功能作用，我们在单周转EPR研究中检测了该自由基氧化AA的能力。在宽单峰自由基形成后立即向ASA-PGHS-2厌氧添加AA，产生了与天然PGHS-2中获得的戊二烯基AA自由基相似的7线EPR信号，或26 - 28 - G单峰自由基。这些EPR信号可分别由戊二烯基自由基和应变烯丙基自由基来解释。使用11d-AA、13(R)d-AA、15d-AA、13,15d(2)-AA和十八氘代AA（d(8)-AA）进行的实验证实，戊二烯基自由基中的未成对电子在C11、C13和C15上离域。当使用16d(2)-AA时观察到6线EPR自由基，表明只有一个强烈相互作用的C16氢。这些结果支持了过氧化物生成的酪氨酰自由基在ASA-PGHS-2的脂氧合酶催化中的功能作用，并且还表明ASA-PGHS-2中的AA自由基比天然PGHS-2中的相应自由基受到更多限制。
