Dataset Information

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

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.

SUBMITTER: Lopata A

PROVIDER: S-EPMC6843921 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Lopata Anna A Jójárt Balázs B Surányi Éva V ÉV Takács Enikő E Bezúr László L Leveles Ibolya I Bendes Ábris Á ÁÁ Viskolcz Béla B Vértessy Beáta G BG Tóth Judit J

Biomolecules 20191017 10

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 EDT ...[more]

PMID: 31627475

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Project description:ObjectiveApproximately 1 in 7 US adults have diabetes; and over 60% of deaths in patients with diabetes have cardiac disease as a principal or contributing cause. Both coronary and peripheral artery disease (PAD) identify high-risk cohorts among patients with diabetes. We have previously demonstrated improved cardiovascular outcomes with edetate disodium-based chelation in post-MI patients with diabetes, enrolled in the Trial to Assess Chelation Therapy (TACT). In these analyses we further studied the effect size of patients with diabetes and severe disease in 2 vascular beds; coronaries, and lower extremity arteries. We questioned whether greater atherosclerotic burden would attenuate the observed beneficial effect of edetate disodium infusions.Research design and methodsThe multicenter TACT used a double blind, placebo controlled, 2 × 2 factorial design with 1708 participants, randomly assigned to receive edetate disodium-based chelation, or placebo and high dose oral vitamins or placebo. There were 162 (9.5% of 1708) post-MI patients with a diagnosis of diabetes mellitus and PAD for this post hoc analysis. Patients received up to 40 double-blind intravenous infusions of edetate disodium-based chelation, or placebo. The composite primary endpoint of TACT consisted of death from any cause, myocardial infarction, stroke, coronary revascularization and hospitalization for angina.ResultsThe median age was 66 years, 15% female, 5% non-Caucasian, and BMI was 31. Insulin was used by 32% of patients. Active infusions significantly reduced the primary endpoint compared with placebo infusions (HR, 0.52; 95% CI, 0.30-0.92; P = 0.0069), with a 30% absolute risk reduction in the primary endpoint. There was a marked reduction in total mortality from 24% to 11%, although of borderline significance (P = 0.052).ConclusionAtherosclerotic disease in multiple vascular beds did not attenuate the beneficial effect of edetate disodium infusions in post MI patients with diabetes. Studies now in progress will prospectively test this post hoc finding.

Dataset Information

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

Publications

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

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