FIG. 4.
Determination of the Km and Vmax for T[S]2. The Kmapp and Vmax for the TR reaction were determined by fitting the Michaelis-Menten equation to the experimental data using nonlinear regression. The kinetic parameters were determined by measuring the TR-catalyzed rate of TNB formation in 50 μl of a buffer containing 40 mM HEPES, 1 mM EDTA, 0.01% BSA, and 0.05% Tween 20. The assay mixture also contained TR (8 mU/ml) and varying T[S]2 concentrations, and the reaction rate was measured over 20 min at 22°C.