FIGURE 2.

AcpA enzyme kinetics in presence of inhibitors. Assays were performed in triplicate in 96-well plates using the model substrate pNPP in 25 mm MES buffer, pH 6.2, with 1 μg/ml purified enzyme. Enzyme activity was monitored continuously, and the initial velocity (V₀) was calculated from the linear range. The kinetic parameters were estimated by nonlinear regression analysis using MicroCal Origin 8. Symbols: The control assay (no inhibitor added) is indicated by ■ in both panels. The symbols used to identify the concentrations of ascorbate are: A, 0.05 mm (□), 0.25 mm (●), 0.5 mm (▴), and 0.75 mm (○). 2-Phosphorascorbate results are depicted in B and the concentrations used were 0.05 μm (□), 0.25 μm (●), 0.5 μm (▴), and 0.75 μm (○). The values used in each figure are the average of three independent determinations. The chemical scaffolds of the inhibitors used in each case are shown as sodium salts.