Format

Send to

Choose Destination
Biomolecules. 2019 Oct 17;9(10). pii: E621. doi: 10.3390/biom9100621.

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity.

Author information

1
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary. lopata.anna@gmail.com.
2
Department of Applied Biotechnology, Budapest University of Technology and Economics, 1111 Budapest, Hungary. lopata.anna@gmail.com.
3
Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, 60438 Frankfurt, Germany. lopata.anna@gmail.com.
4
Institute of Food Engineering, Faculty of Engineering, University of Szeged, 6724 Szeged, Hungary. jojartb@gmail.com.
5
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary. suranyi.eva@ttk.mta.hu.
6
Department of Applied Biotechnology, Budapest University of Technology and Economics, 1111 Budapest, Hungary. suranyi.eva@ttk.mta.hu.
7
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary. enikot@gmail.com.
8
Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, 1111 Budapest, Hungary. bezur@mail.bme.hu.
9
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary. leveles.ibolya@ttk.mta.hu.
10
Department of Applied Biotechnology, Budapest University of Technology and Economics, 1111 Budapest, Hungary. leveles.ibolya@ttk.mta.hu.
11
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary. abris.bendes@oulu.fi.
12
Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, Finland. abris.bendes@oulu.fi.
13
Institute of Chemistry, University of Miskolc, 3515 Miskolc, Hungary. bela.viskolcz@uni-miskolc.hu.
14
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary. vertessy@kutatok.org.
15
Department of Applied Biotechnology, Budapest University of Technology and Economics, 1111 Budapest, Hungary. vertessy@kutatok.org.
16
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1113 Budapest, Hungary. toth.judit@ttk.mta.hu.

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.

KEYWORDS:

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

Supplemental Content

Full text links

Icon for Multidisciplinary Digital Publishing Institute (MDPI) Icon for PubMed Central
Loading ...
Support Center