DHODH enzymatic assay

Enzyme assays were performed with 6 nm recombinant human DHODH prepared as described (36). The reaction mixture for these kinetic assays consisted of 1 mm DHO, 100 μm 2,3-dimethoxy-5-methyl-p-benzoquinone (Sigma–Aldrich #D9150), and 100 μm 2,6-dichlorophenolindophenol sodium salt (DCIP; Sigma–Aldrich #D1878) in enzyme buffer (50 mm Tris-HCl pH 8.0, 0.1% Triton X-100, 150 mm KCl). A stock solution of 20 mm DCIP was prepared in enzyme buffer and filtered through filter paper (20−25-μm pore size) just before use. Loss in absorbance by DCIP was measured at 595 nm at room temperature in a stepped time course (8 × 2 min, 8 × 3 min, 6 × 5 min). The observed decrease in absorbance over time was linear between 8 and 26 min. Therefore, for each concentration of inhibitor tested, a value for DHODH's V_max was estimated by linear regression within this time frame. The IC₅₀ is defined as the concentration of inhibitor that gives V_max([I]) = V_max(DMSO)/2.