Beyond chelation: EDTA tightly binds taq DNA polymerase, MutT and dUTPase and directly inhibits dNTPase activity

Anna Lopata, Balázs Jójárt, Éva V. Surányi, Eniko Takács, László Bezúr, Ibolya Leveles, Ábris Bendes, Béla Viskolcz, Beáta G. Vértessy, Judit Tóth

Research output: Contribution to journalArticle

Abstract

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.

Original languageEnglish
Article number621
JournalBiomolecules
Volume9
Issue number10
DOIs
Publication statusPublished - Oct 2019

Keywords

  • EDTA
  • dNTP hydrolysis
  • dNTP pool sanitizing enzymes
  • dNTPase inhibitor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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