Continuous Kinetic Assay for hDUT Inhibition

Proton release during the hydrolysis of dUTP into dUMP and PP_i was followed continuously at 559 nm at 20 °C using a Specord 200 spectrophotometer (Analytic Jena, Germany) and 10 mm path length thermostatted cuvettes as described previously^{21 (link),42 (link),48 (link)}. Reaction mixtures contained 50 nM hDUT enzyme and varying concentrations of Stl in activity buffer (1 mM HEPES (pH 7.5), 5 mM MgCl₂, 150 mM KCl, 40 μM Phenol Red indicator). The reaction started with the addition of 30 μM dUTP after 5 min pre-incubation of the two proteins in the assay buffer. Initial velocity was determined from the slope of the first 10% of the progress curve. Stl inhibition data were fitted to the quadratic binding equation describing 1:1 stoichiometry for the dissociation equilibrium with no cooperativity^{42 (link)}.