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

Barak Akabayov, Charles C. Richardson

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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 languageEnglish
Pages (from-to)159-162
Number of pages4
JournalPowder Diffraction
Volume26
Issue number2
DOIs
StatePublished - 23 Jun 2011
Externally publishedYes

Keywords

  • DNA polymerase
  • Manganese
  • Metal cofactor
  • Metalloenzyme
  • XAFS

Fingerprint

Dive into the research topics of 'Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7'. Together they form a unique fingerprint.

Cite this