Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition.
Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition.
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DOI：
10.1021/bi9016632
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发表时间：
2010-04-13
期刊：
影响因子：
2.9
通讯作者：
Zweier JL
中科院分区：
文献类型：
--
作者：
Chen W;Druhan LJ;Chen CA;Hemann C;Chen YR;Berka V;Tsai AL;Zweier JL
内皮型一氧化氮合酶(ENOS)是心血管和心脏功能的重要调节因子。过氧亚硝酸盐(ONOO-−)可使eNOS失活，但其作用机制仍存在疑问。据报道，失活是由于eNOS硫代锌簇的氧化，而不是辅因子四氢生物蝶呤(BH4)的氧化；然而，这一点仍然存在很大争议。因此，我们研究了一氧化氮合酶−诱导eNOS功能障碍的机制及其剂量依赖关系。人eNOS暴露于ONOO−后，eNOS二聚体明显失稳，并呈剂量依赖性丧失活性。高效液相分析表明，游离和eNOS结合的BH4在暴露于ONOO−期间都被氧化；然而，蛋白质结合的生物蝶呤完全氧化需要更高的ONOO−水平。此外，ONOO−还引起了酶的紫外可见光谱和血红素含量的变化。内皮型一氧化氮合酶与BH4预孵育可减少二聚体失稳和血红素改变。在ONOO−失稳的eNOS二聚体中加入BH4只能部分挽救酶的功能。与ONOOTPEN处理相比，去除酶结合锌的−处理并未改变eNOS的活性或稳定性，说明BH4诱导的二聚体的稳定不需要锌-硫酸盐簇占据。而ONOO-−处理引起了锌结合的丧失，这不能解释酶活性的丧失。因此，ONOO−诱导的eNOS失活主要是由于BH4的氧化和血红素/血红素中心的不可逆破坏。
Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO−) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH4); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO−-induced eNOS dysfunction and their dose-dependence. Exposure of human eNOS to ONOO− resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH4 were oxidized during exposure to ONOO−; however, full oxidation of protein bound biopterin required higher ONOO− levels. Additionally, ONOO− triggered changes in UV/Visible spectrum and heme content of the enzyme. Pre-incubation of eNOS with BH4 decreased dimer destabilization and heme alteration. Addition of BH4 to the ONOO−-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO− treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH4 does not require zinc occupancy of the zinc-thiolate cluster. While ONOO− treatment was observed to induce loss of Zn-binding this can not account for the loss of enzyme activity. Therefore, ONOO−-induced eNOS inactivation is primarily due to oxidation of BH4 and irreversible destruction of the heme/heme-center.
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