Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg2+, as an example, mediates binding of deoxyribonucleoside 5′-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg2+to an active site because Mg2+is spectroscopically silent and Mg2+binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg2+with Mn2+:Mn2+that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn2+is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn2+that is free in solution and Mn2+bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

中文翻译：

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Mn-脱氧核糖核苷三磷酸与噬菌体 T7 DNA 聚合酶活性位点的结合

二价金属离子作为多种细胞内酶活性的辅助因子是至关重要的。镁2+例如，介导脱氧核糖核苷 5'-三磷酸的结合，然后在 DNA 聚合酶的活性位点水解。很难研究 Mg 的结合2+到一个活跃的网站，因为 Mg2+光谱无声，Mg2+以低亲和力与酶的活性位点结合。因此，我们用 Mg2+含锰2+:锰2+这不仅在光谱上可见，而且还提供了噬菌体 T7 的 DNA 聚合酶的全部活性。为了证明大多数 Mn2+与酶结合，我们使用 X 射线近边缘光谱对 T7 DNA 聚合酶进行定点滴定分析。在这里，我们展示了如何使用 X 射线近边缘光谱来区分源自 Mn 的信号2+在溶液中游离，Mn2+与 T7 DNA 聚合酶的活性位点结合。该方法可以应用于使用二价金属离子作为辅因子的其他酶。