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Native Mass Spectrometry Gives Insight into the Allosteric Binding Mechanism of M2 Pyruvate Kinase to Fructose-1,6-Bisphosphate
Biochemistry ( IF 2.9 ) Pub Date : 2018-03-02 00:00:00 , DOI: 10.1021/acs.biochem.7b01270
Agni F. M. Gavriilidou 1 , Finn P. Holding 2 , Daniel Mayer 3 , Joseph E. Coyle 2 , Dmitry B. Veprintsev 3 , Renato Zenobi 1
Affiliation  

The various oligomeric states of the M2 isoform of pyruvate kinase (PKM2) were distinguished using native mass spectrometry. The effect of PKM2 concentration on its dimer–tetramer equilibrium was monitored, and a value for the dissociation constant (Kd) of the two species was estimated to be 0.95 μM. Results of binding of fructose-1,6-bisphosphate (FBP) to PKM2 are shown and provide insight into the allosteric mechanism and changes in the oligomerization status of PKM2. The average Kd for binding of FBP to the PKM2 tetramer was estimated to be 7.5 μM. It is concluded that four molecules of FBP bind to the active PKM2 tetramer whereas binding of FBP to the PKM2 dimer was not observed. It is suggested that either FBP potentiates rapid tetramer formation after binding to apo PKM2 dimers or FBP binds to PKM2 apo tetramers, thus driving the dimer–tetramer equilibrium in the direction of fully FBP-bound tetramer. The binding occurs in a highly positively cooperative manner with a Hill coefficient (n) of 3.

中文翻译:

天然质谱可深入了解M2丙酮酸激酶与果糖1,6-双磷酸酯的变构结合机理

丙酮酸激酶(PKM2)的M2亚型的各种低聚状态使用天然质谱进行了区分。监测了PKM2浓度对其二聚体-四聚体平衡的影响,两个物种的解离常数(K d)估计为0.95μM。结果显示了果糖1,6-双磷酸酯(FBP)与PKM2的结合结果,可洞悉PKM2的变构机理和低聚状态的变化。平均K dFBP与PKM2四聚体结合的结合力估计为7.5μM。结论是,FBP的四个分子与活性PKM2四聚体结合,而未观察到FBP与PKM2二聚体结合。建议在结合载脂蛋白PKM2二聚体后FBP增强快速四聚体形成,或者FBP结合PKM2载脂蛋白四聚体,从而在完全结合FBP的四聚体方向上驱动二聚体-四聚体平衡。结合以高度正合作的方式发生,希尔系数(n)为3。
更新日期:2018-03-02
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