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Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg 2+ , 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 Mg 2+ to an active site because Mg 2+ is spectroscopically silent and Mg 2+ binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg 2+ with Mn 2+ :Mn 2+ 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 Mn 2+ 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 Mn 2+ that is free in solution and Mn 2+ 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.

Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7