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

Published online by Cambridge University Press:  05 March 2012

Barak Akabayov  and
Charles C. Richardson
Barak Akabayov*
Affiliation:
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Charles C. Richardson*
Affiliation:
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
*
a)Author to whom correspondence should be addressed. Electronic mail: barak_akabayov@hms.harvard.edu
b)Electronic mail: ccr@hms.harvard.edu
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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.

Keywords

DNA polymerasemanganeseXAFSmetal cofactormetalloenzyme
Type
Technical Articles
Information
Powder Diffraction , Volume 26 , Special Issue 2 , June 2011 , pp. 159 - 162
Copyright
Copyright © Cambridge University Press 2011

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