Pyruvate Formate-lyase and Its Activation by Pyruvate Formate-lyase Activating Enzyme
Pyruvate Formate-lyase and Its Activation by Pyruvate Formate-lyase Activating Enzyme
复制标题
DOI：
10.1074/jbc.m113.496877
复制
发表时间：
2014-02-28
影响因子：
4.8
通讯作者：
Broderick, Joan B.
中科院分区：
文献类型：
--
作者：
Crain, Adam V.;Broderick, Joan B.
背景：PFL是一种由自由基谷胱甘肽激活酶（PFL-AE）激活的甘氨酰自由基酶（GRE）。结果：测定了PFL-AE与PFL和TCFMet结合的平衡常数，并定量了底物对活化的影响。结论：在体内，PFL-AE主要以PFL和PAL-Met的复合物形式存在。重要性：GREs在厌氧代谢中起着关键作用，但其激活机制尚不清楚，丙酮酸甲酸裂解酶激活酶（PFL-AE）激活丙酮酸甲酸裂解酶（PFL）需要S-腺苷甲硫氨酸（S-adenosylmethionine，S-Met）参与反应，通过PFL-AE在PFL上形成特异性甘氨酰自由基。在厌氧条件下对氧敏PFL-AE进行表面等离子体共振实验，以确定其与PFL相互作用的动力学和平衡常数。这些实验表明，相互作用是非常缓慢的，并受到大的构象变化的限制。使用一种新的PFL-Met结合测定法来准确测定PFL-AE单独和与PFL复合的PFL-Met结合的平衡常数。PFL-AE以相同的亲和力（约6 μ m）结合C3 Met，而与PFL的存在与否无关。PFL在其底物丙酮酸或类似物草氨酸存在下的活化导致[4Fe-4S]（1+）簇化学计量转化为PFL上的甘氨酰自由基;然而，在不存在这些小分子的情况下实现了3.7倍的活化，表明丙酮酸或草氨酸是最佳活化所需的。最后，计算整个PFL系统的体内浓度以估计细胞中结合蛋白的量。PFL，PFL-AE，和C3 Met在体内基本上完全结合，而电子供体蛋白质部分结合。
Background: PFL is a glycyl radical enzyme (GRE) activated by a radical AdoMet-activating enzyme (PFL-AE). Results: Equilibrium constants for PFL-AE binding to PFL and AdoMet are determined, and the effects of substrates on activation are quantified. Conclusion:In vivo, PFL-AE exists largely in complex with PFL and AdoMet. Significance: GREs play key roles in anaerobic metabolism, but their activation is poorly understood.The activation of pyruvate formate-lyase (PFL) by pyruvate formate-lyase activating enzyme (PFL-AE) involves formation of a specific glycyl radical on PFL by the PFL-AE in a reaction requiring S-adenosylmethionine (AdoMet). Surface plasmon resonance experiments were performed under anaerobic conditions on the oxygen-sensitive PFL-AE to determine the kinetics and equilibrium constant for its interaction with PFL. These experiments show that the interaction is very slow and rate-limited by large conformational changes. A novel AdoMet binding assay was used to accurately determine the equilibrium constants for AdoMet binding to PFL-AE alone and in complex with PFL. The PFL-AE bound AdoMet with the same affinity (approximate to 6 m) regardless of the presence or absence of PFL. Activation of PFL in the presence of its substrate pyruvate or the analog oxamate resulted in stoichiometric conversion of the [4Fe-4S](1+) cluster to the glycyl radical on PFL; however, 3.7-fold less activation was achieved in the absence of these small molecules, demonstrating that pyruvate or oxamate are required for optimal activation. Finally, in vivo concentrations of the entire PFL system were calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and AdoMet are essentially fully bound in vivo, whereas electron donor proteins are partially bound.