Dicing Assays

Fiji software was used to quantify the substrate and product bands in the raw image file and to correct for background. To determine IC₅₀ values in substrate competition assays, product formed as a function of competitor concentrations was fit to a three-parameter inhibition model:

To determine rates of reaction, product formed as a function of time was fit to the burst-and-steady-state equation:

The time-dependence of product formation corresponded to a pre-steady-state exponential burst (k_burst = k₂ + k₃) followed by a linear steady-state phase, described by k_cat, where k_cat = k₂ × k₃/(k₂ + k₃). The size of the burst corresponds to concentration of active enzyme, E_active.