Nitro-fatty acid reaction with glutathione and cysteine - Kinetic analysis of thiol alkylation by a Michael addition reaction
Nitro-fatty acid reaction with glutathione and cysteine - Kinetic analysis of thiol alkylation by a Michael addition reaction
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DOI：
10.1074/jbc.m704085200
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发表时间：
2007-10-19
影响因子：
4.8
通讯作者：
Freeman, Bruce A.
中科院分区：
文献类型：
--
作者：
Baker, Laura M. S.;Baker, Paul R. S.;Freeman, Bruce A.
一氧化氮衍生的物质的脂肪酸硝化产生亲电产物，其加合蛋白质硫醇，诱导蛋白质功能和分布的变化。在健康人血液中检测到蛋白质和还原型谷胱甘肽（GSH）的硝基脂肪酸加合物。动力学和质谱分析表明，油酸（OA-NO2）和亚油酸（LNO 2）的硝基烯烃衍生物与GSH和Cys通过Michael加成反应快速反应。通过停流分光光度法测定的OA-NO2和LNO 2与GSH反应的速率显示，在pH 7.4和37 ℃下，二级速率常数分别为183 M-1 s（-1）和355 M（-1）s（-1）。这些反应速率明显大于GSH与过氧化氢和非硝化亲电脂肪酸（包括8-异前列腺素A（2）和15-脱氧-δ（12，14）-前列腺素J（2））的反应速率。增加反应pH值从7.4到8.9增强的表观二阶速率常数与OA-NO2和LNO 2的硫醇反应，表现出依赖于GSH的硫醇阴离子的反应性。硝基烯烃与硫醇的反应速率随着目标硫醇的pK（a）的增加而降低。增加浓度的洗涤剂辛基-β-D-吡喃葡萄糖苷降低了硝基烯烃与GSH的反应速率，表明硝基脂肪酸组织成胶束或膜结构可以限制与更极性的亲核目标的Michael反应性。总之，这些结果表明，硫醇的可逆加合硝基脂肪酸是一种机制，可逆的蛋白质功能的硝基脂肪酸的翻译后调节。
Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M-1 s(-1) and 355 M(-1)s(-1), respectively, at pH 7.4 and 37 degrees C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A(2) and 15-deoxy-Delta(12,14)-prostaglandin J(2). Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pK(a) of target thiols increased. Increasing concentrations of the detergent octyl-beta-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.