Figure 3. Selection of optimum incubation time and enzyme concentration for kinetic parameter assay.

To determine the scope of initial reaction velocity, enzymatic reactions (100 µl) containing 0.5 mM alanine, 0.5 mM ATP and different concentrations of purified Sare0718 were performed at 25°C in 96-well plates for various times (2 min, 5 min, 10 min, and 15 min). (A) Enzyme concentration curve: reaction velocity versus concentration of Sare0718. (B) Time course curve: concentration of production PPi versus reaction time. Sare0718 concentrations in curve 1–6 were 0.0625 µM, 0.125 µM, 0.25 µM, 0.375 µM, 0.5 µM and 0.625 µM, respectively.