Enzymatic activity and inhibition assay

The activity and inhibition of E. coli enolase described in this study was carried out using the procedure as we previously reported⁶. Briefly, enolase activity was measured at 25 °C in the forward (formation of PEP from 2‐PGA) direction by direct monitoring of the increase in PEP absorbance at 240 nm, using a DU 640 spectrophotometer (Beckman). The standard assay contained 50 mM Tris pH 8.0, 0.1 M KCl, 0.5 mM 2-PGA and 1 mM MgSO₄. The reactions were carried out at a final volume of 500 μL. Initial velocities came from the slopes of linear progress curves of 1 minute duration. One unit of enolase activity was defined as the amount of protein which catalyzes the formation of 1 μmole PEP from 2-PGA in 1 minute. The concentration of PEP was determined using a molar extinction coefficient (Ɛ_240nm = 1400 M⁻¹ cm¹).

Inhibition of enolase activity by SF2312 and its analog, KSF was performed at a constant enolase concentration of 40 nM. The compounds, supplied in water, were preincubated with protein at concentrations ranging from 2 nM to 20 µM for 5 minutes in the assay buffer consisting of 50 mM Tris pH 8.0, 0.1 M KCl and 1 mM MgSO₄. The reaction was initiated by the addition of 0.5 mM 2-PGA. The decrease in enolase activity upon inhibition was monitored at 5 second intervals for a period of 5 minutes at λ =240 nm. The average half maximal inhibitory concentrations (IC₅₀) was calculated from initial rates of absorbance increase plotted as a function of ligand concentration using GraphPad Prism software (Fig. 2). K_i values were calculated using the Cheng-Prusoff equation from the experimental IC₅₀ and K_m values.