Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, U.K.
Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, U.K.
Biochem J. 2018 Oct 31;475(20):3275-3291. doi: 10.1042/BCJ20180556.
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 studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the 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.
我们在此表明，人丙酮酸激酶的 M2 同工型（M2PYK）易发生亚硝化和氧化，并且这些修饰通过防止活性四聚体形式的形成来调节酶活性。对 M1 和 M2 同工型进行的生物素转换测定表明，M2PYK 易发生亚硝化，并且 Cys326 极易发生氧化还原修饰。对三个半胱氨酸残基（Cys424、Cys358 和 Cys326）的点突变进行了结构和酶学研究，以表征它们在氧化还原调节中的潜在作用。在 M2PYK 和 M1PYK 之间有 9 个半胱氨酸保守。Cys424 是 M2PYK 独有的半胱氨酸。与 M2PYK 相比，C424S、C424A 和 C424L 对酶活性的影响适中，分别为 80%、100%和 140%。C358 先前已从研究中被确定为氧化的首选靶标。我们表征的突变体表明，该突变稳定了四聚体 M2PYK，这表明 Cys358Ser 突变对氧化的抗性是由于酶的四聚体形式的稳定。相比之下，Cys326Ser 突变体主要以单体形式存在。使用该突变体进行的生物素转换测定也显示 M2PYK 的生物素化显著减少，证实这是亚硝化和可能氧化的主要靶标。我们的结果表明，M2PYK 对氧化和亚硝化的敏感性受其单体-四聚体平衡调节。在单体状态下，残基（特别是 C326）易发生氧化修饰，从而阻止活性四聚体形式的重新形成。
