Prostaglandin H synthase: spectroscopic studies of the interaction with hydroperoxides and with indomethacin.
Prostaglandin H synthase: spectroscopic studies of the interaction with hydroperoxides and with indomethacin.
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前列腺素 H 合酶：与氢过氧化物和吲哚美辛相互作用的光谱研究。
DOI：
10.1021/bi00489a037
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Palmer,G
中科院分区：
文献类型：
--
作者：
Kulmacz,RJ;Ren,Y;Tsai,AL;Palmer,G
Department of Biochemistry and Cell Biology，Rice University，Houston，Texas 77251 Received January 29，1990; Revised Mandarin pt Received May 8，1990摘要：前列腺素H合酶具有血红素依赖性过氧化物酶活性和环加氧酶活性。目前的假说认为环加氧酶反应是自由基链式反应，由合成酶过氧化物酶与氢过氧化物的相互作用引发，导致酪氨酰自由基的产生[Stubbe，J.A.（1989）Annu. Rev.Biochem.58，257-285]。我们已经研究了自由基形成与乙基过氧化氢（EtOOH）和15-hydroperoxyeicosatetraenoic酸（15-HPETE）的动力学，并分析了吲哚美辛（一种选择性的环氧合酶抑制剂）和四硝基甲烷（TNM;选择剂硝化酪氨酰残基）的合成酶的影响。在-14 ℃下，EtOOH和15-HPETE均在5 s内产生自由基物质，其电子顺磁共振谱由中心在g=2.005处的双峰（约16 G的分裂; 35 G的总峰-谷宽度）主导，其归因于酪氨酰基自由基。双峰随后让位于具有相似峰谷宽度的单峰;双峰到单峰的转变在20-60秒内完成。在使用EtOOH 120 s后，双峰/单峰组合的强度达到峰值，为0.6自旋/血红素，在使用15-HPETE 20 s后达到约0.3自旋/血红素;使用EtOOH时，自由基强度下降缓慢，但使用15-HPETE时下降更快。吲哚美辛合酶复合物与EtOOH的反应导致较窄的（峰谷宽度为24 G）单线态自由基信号，没有早期双峰的证据;单线态的强度在约300 s后达到峰值0.45自旋/血红素。TNM处理的合成酶与EtOOH的反应产生了与吲哚美辛合成酶复合物几乎相同的单线态。在pH 8.0的合成酶全酶与TNM的反应导致环氧合酶和过氧化物酶活性的失活，前者被迅速和完全失去，而后者损失缓慢，约50%。竞争性环氧合酶抑制剂Iceland使环氧合酶的失活速率减慢约20倍。TNM对合酶脱辅基酶中环氧合酶活性的失活率也比全酶低约20倍。氨基酸分析表明，TNM反应全酶的残余活性< 10%，含有1.8个硝基酪氨酸/亚基;脱辅基酶在相同条件下反应，具有> 80%的原始活性，含有0.7个硝基酪氨酸/亚基。合酶与过氧化氢的反应，从而出现导致两个酪氨酰基自由基的顺序生成，其中一个或两个自由基所需的环氧合酶催化。电子吸收和磁性圆二色光谱的合成酶全酶之前和之后，另外的血红素配体（氰化物，叠氮化物，和氟化物）和电子顺磁光谱的休息合成酶的吲哚美辛合成酶复合物的相应的光谱变化不大。因此，吲哚美辛似乎并没有显著改变合酶中的血红素环境;这种药物对环氧合酶的抑制更可能是由于对过氧化氢诱导的自由基的干扰。Rostaglandins H合酶催化一系列生理上重要的化合物，即前列腺素、血栓烷和前列环素的生物合成中的第一个关键步骤。该酶具有两种不同的催化活性：将花生四烯酸转化为前列腺素G2的环氧合酶。
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251 Received January 29, 1990; Revised Manuscript Received May 8, 1990 abstract: Prostaglandin H synthase has both a heme-dependent peroxidase activity and a cyclooxygenase activity. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by an interaction of thesynthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical [Stubbe, J. A.(1989) Annu. Rev. Biochem. 58, 257-285]. We have examined the kinetics of radical formation with both ethyl hydroperoxide (EtOOH) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and have analyzed the effects of indomethacin (a selective cyclooxygenase inhibitor) and tetranitromethane (TNM; a selective agent for nitration of tyrosyl residues) on the synthase. At-14 C both EtOOH and 15-HPETE generated within 5 s a free radical species whose electron paramagnetic resonance spectrum was dominated by a doublet centered at g=2.005 (splitting of~ 16 G; overall peak-to-trough width of 35 G) that has been attributed to a tyrosyl radical. The doublet subsequently gave way to a singlet with a similar peak-to-trough width; the doublet-to-singlet transition was complete in 20-60 s. The intensity of the doublet/singlet combination peaked at 0.6 spins/heme after 120 s with EtOOH and at about 0.3 spins/heme after 20 s with 15-HPETE; the radical intensity declined slowlywith EtOOH but more rapidly with 15-HPETE. Reaction of the indomethacin-synthase complex with EtOOH resulted in a narrower(peak-to-trough width of 24 G) singlet free radical signal, with no evidence of an earlier doublet; the intensity of the singlet peaked at 0.45 spins/heme after about 300 s. Reaction of TNM-treated synthase with EtOOH resulted in a singlet almost identicalwith that seen for the indomethacin-synthase complex. Reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both cyclooxygenase and peroxidase activity, with the former being lost rapidly and completely while the latter was lost slowly and to about 50%. Ibuprofen, a competitive cyclooxygenase inhibitor, slowed the rate of inactivation of the cyclooxygenase by about 20-fold. The rate of inactivation of the cyclooxygenase activity in synthase apoenzyme by TNM was also about20-fold less than that observed with the holoenzyme. Amino acid analyses revealed that TNM-reacted holoenzyme with< 10% residual activity contained 1.8 nitrotyrosines/subunit; apoenzyme reacted under the same conditions had> 80% of the original activity and contained 0.7 nitrotyrosine/subunit. Reaction of the synthase with hydroperoxides thus appears to lead to the sequential generation of two tyrosyl radicals, with one or both of the radicals requiredfor cyclooxygenase catalysis. The electronic absorbance and magnetic circular dichroism spectra of the synthase holoenzyme before and after addition of heme ligands (cyanide, azide, and fluoride) and the electron paramagnetic spectrum of the resting synthase were little changed from the corresponding spectra of the indomethacin-synthase complex. Indomethacin thus does notappear to greatlyalter the heme environment in the synthase; inhibition of the cyclooxygenase by this agent is more likely due to a perturbation of the hydroperoxide-inducedradical species.-Rostaglandin H synthase catalyzes the first committed step in the biosynthesis of a series of physiologically important compounds, namely prostaglandins, thromboxanes, and prostacyclins. The synthasehas two distinct catalytic activities: a cyclooxygenase that converts arachidonic acid to prostaglandin G2 …
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DOI：
10.1016/s0021-9258(18)95676-0
发表时间：
1985-12
期刊：
Advances in prostaglandin, thromboxane, and leukotriene research
影响因子：
--
作者：
A. Lambeir;C. Markey;H. Dunford;L. Marnett
通讯作者：
A. Lambeir;C. Markey;H. Dunford;L. Marnett
肌红蛋白复合物的磁圆二色性研究。
影响因子：
15
作者：
L. Vickery;T. Nozawa;K. Sauer
通讯作者：
K. Sauer
DOI：
--
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Kulmacz,RJ;Lands,WE
通讯作者：
Lands,WE
影响因子：
2.9
作者：
M. Sokolovsky;J. Riordan;B. Vallée
通讯作者：
B. Vallée
前列腺素 H 合酶中血红素自旋态和过氧化物诱导的自由基种类。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Kulmacz,RJ;Tsai,AL;Palmer,G
通讯作者：
Palmer,G