4.2.1. MbtI Enzymatic Assays

Recombinant M. tuberculosis MbtI was produced and purified as previously reported [14]. Enzyme activity was determined at 37 °C, measuring the formation of salicylic acid by a fluorimetric assay, slightly modified from Vasan et al. [38]. Briefly, the reactions were performed in a final volume of 400 μL of 50 mM Hepes pH 7.5, 5 mM MgCl₂, containing 1-2 μM MbtI, by the addition of chorismic acid, and monitored using a Perkin-Elmer LS3 fluorimeter (Ex. λ = 305 nm, Em. λ = 420 nm). Inhibition assays were performed in the presence of the compound at 100 µM (stock solution 20 mM in DMSO) and 50 µM chorismic acid. Where possible, compounds were tested both as free acids and sodium salts, providing analogous results. For compounds inhibiting by more than 75% the initial activity, IC₅₀ values were determined. To this end, the activity was measured at different compound concentrations, and values were calculated according to Equation (1), with Origin 8 software:

where A_[I] is the activity at inhibitor concentration [I] and A[0] is the activity in the absence of the inhibitor.

The K_i was determined at different substrate [S] and compound concentrations, using Equation (2):