Enzyme assays.

IMPDH assays were performed in 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 3 mM EDTA, and 1 mM DTT. Activity was routinely assayed in the presence of 20 to 50 nM IMPDH at 25°C. NADH production was monitored either by following the absorbance change at 340 nm, using a Hitachi U-2000 spectrophotometer (ε = 6.2 mM⁻¹ cm⁻¹), or by following fluorescence on a Biotek plate reader (excitation wavelength = 340 nm; emission wavelength = 460 nm). Each determination of K_i,app was derived from duplicate measurements of enzyme activity in the presence of 12 different inhibitor concentrations. Values reported in the tables are averages for at least two independent determinations. Enzyme was incubated with inhibitor (50 pM to 100 μM) for 10 min at room temperature prior to addition of substrates. The concentrations of IMP and NAD⁺ were fixed at 1.0 mM (saturating) and 0.9 mM (2.4× K_m), respectively. K_i,app values were calculated for each inhibitor according to either equation 1 or equation 2, using the SigmaPlot program (SPSS, Inc.). Equation 1 was as follows:

where v_i is the initial velocity in the presence of inhibitor (I) and v₀ is the initial velocity in the absence of inhibitor. The Morrison equation (equation 2) was used to evaluate tight-binding inhibitors (55), as follows:

where v_i is the initial velocity in the presence of inhibitor, v₀ is the initial velocity in the absence of inhibitor, E₀ is the total concentration of enzyme, I₀ is the total concentration of inhibitor, and K* is the apparent inhibition constant.