TY  - JOUR
A1  - Lopata, Anna
A1  - Jójárt, Balázs
A1  - Surányi, Éva V.
A1  - Takács, Enikő
A1  - Bezúr, László
A1  - Leveles, Ibolya
A1  - Bendes, Ábris Á.
A1  - Viskolcz, Beĺa
A1  - Vértessy, Beáta G.
A1  - Tóth, Judit
T1  - Beyond chelation: EDTA tightly binds taq DNA polymerase, MutT and dUTPase and directly inhibits dNTPase activity
T2  - Biomolecules
N2  - 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.
KW  - dNTP hydrolysis
KW  - EDTA
KW  - dNTP pool sanitizing enzymes
KW  - dNTPase inhibitor
Y1  - 2019
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/52569
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-525690
SN  - 2218-273X
N1  - This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
VL  - 9
IS  - 10, Art. 621
SP  - 1
EP  - 19
PB  - MDPI
CY  - Basel
ER  -