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首页> 外文期刊>Biochemistry >Role of Metal Ions in Catalysis by Enolase: An Ordered Kinetic Mechanism for a Single Substrate Enzyme
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Role of Metal Ions in Catalysis by Enolase: An Ordered Kinetic Mechanism for a Single Substrate Enzyme

机译：金属离子在烯醇酶催化中的作用：单一底物酶的有序动力学机制。

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摘要

Spectroscopic and kinetic methods have been used to explore the roles of divalent metal ions in the enolase-catalyzed dehydration of 2-phosphoglycerate (2-PGA). Enolase requires 2 equiv of metal ion per active site for maximal activity. Previous crystallographic studies [Larsen, T. M., Wedekind, I. E., Rayment, I., and Reed, G. H.(1996) Biochemistry 35,4349-4358] showed that both magnesium ions coordinated to the carboxylate group of the substrate/product-a scheme consistent with metal ion assistance in formation of the enolate intermediate. Electron paramagnetic resonance (EPR) data with 170-labeled forms of phosphoenolpyruvate show that Mn2+ , bound at the lower affinity site, coordinates to one carboxylate oxygen and one phosphate oxygen of the substrate. These observations are fully consistent with the crystallographic data. Plots of activity versus log [metal ion] are bell-shaped, and the inhibitory phases of the profiles have been previously attributed to binding of metal ions at ancillary sites on the enzyme. However, the activation profiles and measurements of2H kinetic isotope effects support an ordered kinetic mechanism wherein binding of2-PGA precedes binding of the second metal ion, and release of the second metal ion occurs prior to departure of phosphoenolpyruvate. High concentrations of metal ion lead to inhibition in the ordered mechanism by interfering with product release. The 2H kinetic isotope effect is diminished in the inhibitory phases of the metal ion activation profiles in a manner that is consistent with the predominantly ordered mechanism. Zn~(2+) gives lower maximal activity than Mg~(2+) , apparently due to slow release of Zn2+ from the product complex. Addition of imidazole increases the maximal rate apparently by accelerating the release of Zn~(2+) from the enzyme.
机译：光谱和动力学方法已用于探索二价金属离子在烯醇酶催化的2-磷酸甘油酸酯（2-PGA）脱水中的作用。烯醇化酶每个活性位点需要2当量的金属离子，以发挥最大活性。以前的晶体学研究[Larsen，TM，Wedekind，IE，Rayment，I.和Reed，GH（1996）Biochemistry 35,4349-4358]显示，两种镁离子均与底物/产物的羧酸根基团配位-方案一致与金属离子协助形成烯醇化物中间体。具有170种标记形式的磷酸烯醇丙酮酸的电子顺磁共振（EPR）数据显示，结合在较低亲和力位点上的Mn2 +与底物的一种羧酸氧和一种磷酸氧配位。这些观察结果与晶体学数据完全一致。活性与对数[金属离子]的关系图呈钟形，曲线的抑制相先前已归因于金属离子在酶辅助位点上的结合。然而，2 H动力学同位素效应的活化曲线和测量结果支持有序的动力学机理，其中2-PGA的结合先于第二金属离子的结合，并且第二金属离子的释放发生在磷酸烯醇丙酮酸的离开之前。高浓度的金属离子通过干扰产物释放而导致有序机理的抑制。 2H动力学同位素效应在金属离子活化曲线的抑制相中以与主要有序机制相一致的方式减弱。 Zn〜（2+）的最大活性低于Mg〜（2+），这显然是由于Zn2 +从产物配合物中的缓慢释放所致。咪唑的添加通过加速酶中Zn〜（2+）的释放而明显提高了最大速率。

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