FIGURE 1.

Effect of small molecules on AcpA phosphatase activity. Enzyme activity was measured using 1 mm pNPP as substrate in 25 mm MES, pH 6.2, buffer. A control assay without inhibitor was always included in the plates as positive internal standard. The development of yellow color was quantified by continuous reading at 412 nm, and the p-nitrophenol released by the action of the enzyme was calculated using ϵ = 17.7 mm⁻¹ cm⁻¹. Screening was performed in 96-well format in a final volume of 300 μl with 1 μg/ml of reaction mixture of purified enzyme. The enzyme activity was assayed against 1152 small molecules dispensed in the Prestwick Chemical Library. The effects of ascorbate on AcpA activity, indicated by the arrowhead, inhibited >40% the release of p-nitrophenol and was selected for further studies.