Nitric oxide inhibits methionine synthase activity in vivo and disrupts carbon flow through the folate pathway
Nitric oxide inhibits methionine synthase activity in vivo and disrupts carbon flow through the folate pathway
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DOI：
10.1074/jbc.m104043200
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发表时间：
2001-07-20
影响因子：
4.8
通讯作者：
Boss, GR
中科院分区：
文献类型：
--
作者：
Danishpajooh, IO;Gudi, T;Boss, GR
一氧化氮的许多生物学效应是通过一氧化氮与含血红素的蛋白质中的铁结合来实现的。钴胺(维生素B-12)在结构上类似于血红素，是叶酸代谢的关键酶蛋氨酸合成酶的辅因子，NO在体外抑制蛋氨酸合成酶的活性，但关于NO与钴胺结合的数据存在争议。我们现在的光谱显示，NO与钴胺的所有三个价态都发生反应，并且NO对蛋氨酸合成酶活性的抑制很可能与其与单价钴胺的反应有关。通过将[C-14]甲基四氢叶酸的甲基部分掺入蛋白质中，我们在培养的哺乳动物细胞中证明了NO抑制蛋氨酸合成酶的活性。甲硫氨酸合成酶活性的抑制扰乱了通过叶酸途径的碳流，通过减少[C-14]甲酸盐掺入蛋氨酸、丝氨酸和嘌呤核苷酸、同型半胱氨酸，但不包括半胱氨酸，减弱了NO对嘌呤合成的抑制，进一步证明了NO是通过抑制蛋氨酸合成酶起作用的。在共培养实验中，无论是在细胞中不加入供体时，还是在生理上产生NO时，都观察到了NO的作用。用MO合成酶抑制剂处理细胞可增加甲酸盐对蛋氨酸、丝氨酸和嘌呤的掺入，以及甲基四氢叶酸对蛋白质的掺入。因此，生理浓度的NO似乎通过叶酸途径调节碳缺陷。
Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B-12) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism, NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin, By following incorporation of the methyl moiety of [C-14]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [C-14]formate into methionine, serine, and purine nucleotides, Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an MO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flaw through the folate pathway.