Abstract
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.
Original language | American English |
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Pages (from-to) | 159-162 |
Number of pages | 4 |
Journal | Powder Diffraction |
Volume | 26 |
Issue number | 2 |
DOIs | |
State | Published - 23 Jun 2011 |
Externally published | Yes |
Keywords
- DNA polymerase
- Manganese
- Metal cofactor
- Metalloenzyme
- XAFS
All Science Journal Classification (ASJC) codes
- Condensed Matter Physics
- Instrumentation
- Radiation
- General Materials Science