Fig 4. Determination of optimal TPR1e triphosphorylation conditions.

(A) Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl₂, and 50 mM Tris/HCl pH 8.3 (B) Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl₂ over Tmp. (C) Influence of the free Mg²⁺ concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg²⁺ concentration is the total Mg²⁺ concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg²⁺ at these concentrations and pH 8.3 [16]. The grey arrows indicate the optimum condition for each series of experiments. Note that the scale in (A) is different from the scale in (B) and (C). Error bars are standard deviations of triplicate experiments.