University of Oulu

Lopata, A.; Jójárt, B.; Surányi, É.V.; Takács, E.; Bezúr, L.; Leveles, I.; Bendes, Á.Á.; Viskolcz, B.; Vértessy, B.G.; Tóth, J. Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity. Biomolecules 2019, 9, 621.

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

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Author: Lopata, Anna1,2,3; Jójárt, Balázs4; Surányi, Éva V.1,2;
Organizations: 1Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary
2Department of Applied Biotechnology, Budapest University of Technology and Economics, 1111 Budapest, Hungary
3Institute of Biophysical Chemistry, Goethe University, 60438 Frankfurt am Main, Germany
4Institute of Food Engineering, Faculty of Engineering, University of Szeged, 6724 Szeged, Hungary
5Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, 1111 Budapest, Hungary
6Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, Finland
7Institute of Chemistry, University of Miskolc, 3515 Miskolc, Hungary
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 4.3 MB)
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Language: English
Published: Multidisciplinary Digital Publishing Institute, 2019
Publish Date: 2020-04-02


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.

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Series: Biomolecules
ISSN: 2218-273X
ISSN-E: 2218-273X
ISSN-L: 2218-273X
Volume: 9
Issue: 10
Article number: 621
DOI: 10.3390/biom9100621
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Funding: This work was supported by the National Research, Development and Innovation Office [K115993, FK124527, K119493, NVKP_16-1-2016-0020, 2017-1.3.1-VKE-2017-00002, 2017-1.3.1-VKE-2017-00013, VEKOP-2.3.2-16-2017-00013 to BGV, NKP-2018-1.2.1-NKP-2018-00005] and by the BME-Biotechnology FIKP grant (BME FIKP-BIO) of the Ministry of Human Capacities. AL was awarded the ‘Students for science’ grant of the Sándor Wekerle Foundation. BV thanks for the financial support for the European Union and the Hungarian State, co-financed by the European Regional Development Fund in the framework of the GINOP-2.3.4-15-2016-00004 project, aimed to promote the cooperation between the higher education and the industry. JT is the recipient of the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Funding for open access charge: Hungarian Scientific Research Funds.
Copyright information: © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (