Product inhibition kinetics determinations - Substrate interaction affinity and enzymatic kinetics using one quantitative FRET assay

Product inhibition is a common phenomenon during enzyme-catalyzed reactions. Almost all product molecules of an enzyme reaction should have some structural similarities to the substrate, and can thus still have affinities to the active site of the enzyme as product inhibitor. Currently, the characterizations of product inhibition are generally carried out by different methods to determine product binding affinity to the enzyme and the enzyme kinetics parameters, and then these parameters are combined to determine product inhibition. However, due to different sensitivity and variations, kinetics parameters determined from different methods are often not compatible, resulting in not accurate measurement. Here, we report a novel method that determines the two different classes of kinetics parameters, IC50 and Ki(or KD), Kcat and KM, using one single assay method-quantitative FRET(qFRET) assay for characterizing the product inhibition of pre-SUMO1's maturation by its protease SENP1. One method to determine all kinetics parameters provides, for the first time, not only a convenient method to determine all kinetics parameters, but more importantly, a novel approach to combine different measurements with mutually compatible results and errors.