Faller L D, Baroudy B M, Johnson A M, Ewall R X
Biochemistry. 1977 Aug 23;16(17):3864-9. doi: 10.1021/bi00636a023.
It has generally been concluded that two divalent cations are required for enolase activity, even though the enzyme is a homodimer that specifically binds four metal ions in the presence of substrate. This paper reports a reinvestigation of the stoichiometry of enolase activation. Specific ion electrode measurements of Mg2+ binding in the presence and absence of substrate are compared with stopped-flow measurements of the velocity of 2-phosphoglycerate dehydration. It is concluded that the enzyme is inactive when only two metal-binding sites are filled and that four sites must be populated with Mg2+ for full activity. An ordered binding mechanism is proposed that quantitatively predicts the activation of enolase by the four Mg2+ ions from their measured dissociation constants and the Michaelis constant for the dehydration reaction. To explain the loss of enzymatic activity at still higher metal concentrations, the binding of additional, inhibitory Mg2+ ions is postulated.
一般认为，烯醇化酶的活性需要两个二价阳离子，尽管该酶是一种同型二聚体，在底物存在的情况下能特异性结合四个金属离子。本文报道了对烯醇化酶激活化学计量学的重新研究。将在有底物和无底物情况下Mg2+结合的特定离子电极测量结果与2-磷酸甘油酸脱水速度的停流测量结果进行了比较。得出的结论是，当只有两个金属结合位点被占据时，该酶无活性，而四个位点都必须被Mg2+占据才能达到完全活性。提出了一种有序结合机制，该机制从测得的解离常数和脱水反应的米氏常数出发，定量预测了四个Mg2+离子对烯醇化酶的激活作用。为了解释在更高金属浓度下酶活性的丧失，假定存在额外的抑制性Mg2+离子的结合。
