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Biology Articles » Biochemistry » Enzymology » Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate » Figures

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- Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

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Figure 1. Chromatograms showing the synthesis of AThTP (arrow) in the high molecular mass fraction (Sephadex G-200, peak I). The incubation was carried out for 1 h under the conditions described in the Methods secion in the absence (a) and the presence (b) of 1 mM ADP. The flow rate was 0.5 ml/min.

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Figure 2. Time dependence of AThTP synthesis. The experiment was carried out under standard conditions (1 mM ADP, 0.1 mM ThDP, 10 mM Mg²⁺, maleate buffer, pH 6.5), and the protein concentration was 100 μg/ml.

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Figure 3. Effect of pH on AThTP synthesizing enzyme. The Mg²⁺concentration was 10 mM, and the buffers used were as follows: (○) 50 mM acetate, pH 5.5; 50 mM maleate, pH 6.0–6.5; 50 mM Tris-maleate, pH 7.0; 50 mM Tris-HCl, pH 7.5; (■) 50 mM Bis-Tris-propane; (□) 100 mM Bis-Tris-propane.

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Figure 4. Effect of ThDP concentration on the activity of AThTP-synthesizing enzyme at fixed concentrations of ADP (1 mM) and Mg²⁺(10 mM). The continuous line was obtained by nonlinear regression using the Michaelis-Menten equation with an apparent K_mof 4.2 ± 0.5 mM. Inset shows the Hanes plot of the data and the line was obtained by linear regression. The results are expressed as mean ± SD for 3 experiments.

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Figure 5. Effect of varying ADP concentrations on the activity of AThTP-synthesizing enzyme at a fixed ThDP concentration of 0.1 mM and 10 mM Mg²⁺. The results are the mean ± SD for 3 to 6 experiments. The line was obtained by nonlinear regression assuming a sigmoid dose-response curve with an EC₅₀of 0.08 mM. The inset shows a Hill plot obtained for ADP concentrations ranging from 0.04 to 0.6 mM. The Hill coefficient (n_H= 2.1) was calculated from the slope of the regression line over the linear portion of the graph (0.01 – 0.20 mM ADP).

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Figure 6. Effect of Mg²⁺concentration on the activity of AThTP-synthesizing enzyme at a fixed ThDP concentration of 0.1 mM and 1 mM ADP. Total Mg²⁺concentration (○); free Mg²⁺concentration (●).