Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.
Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.
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DOI：
10.1042/bcj20180556
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发表时间：
2018-10-31
期刊：
影响因子：
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通讯作者：
Walkinshaw MD
中科院分区：
文献类型：
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作者：
Mitchell AR;Yuan M;Morgan HP;McNae IW;Blackburn EA;Le Bihan T;Homem RA;Yu M;Loake GJ;Michels PA;Wear MA;Walkinshaw MD
我们在这里表明，M2亚型的人丙酮酸激酶（M2 PYK）是易受亚硝化和氧化，这些修改调节酶的活性，防止形成的活性四聚体形式。对M1和M2亚型进行的生物素开关测定表明，M2 PYK对亚硝化敏感，Cys 326对氧化还原修饰高度敏感。结构和酶的研究已经进行了点突变的三个半胱氨酸残基（Cys 424，Cys 358，和Cys 326），以确定其潜在的作用，在氧化还原调节。在M2 PYK和M1 PYK之间有9个半胱氨酸是保守的。Cys 424是M2 PYK唯一的半胱氨酸。与M2 PYK相比，C424 S、C424 A和C424 L对酶活性表现出中等影响，分别为80、100和140%的活性。先前已从体内研究中确定C358是氧化的有利靶点。我们表征的突变体表明，该突变稳定四聚体M2 PYK，表明Cys 358 Ser突变的体内抗氧化性是由于酶的四聚体形式的稳定。相反，Cys 326 Ser突变体主要以单体形式存在。使用该突变体的生物素开关测定也显示M2 PYK的生物素化显著降低，证实这是亚硝化和可能氧化的主要靶标。我们的研究结果表明，M2 PYK的氧化和亚硝化的敏感性是由它的单体-四聚体平衡。在单体状态下，残基（特别是C326）暴露于氧化修饰，防止活性四聚体形式的重组。
We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer–tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.