Single time-point analysis of product and substrate inhibition

When enzyme inhibition by either the product or excess substrate occurs, it is possible to determine the characteristic kinetic parameters based on [P]/t measurements, even when a large proportion of the substrate is converted. The advantages of various approaches are discussed. Most of them allow a good estimation of the V and K_m values. Conversely, the determination of K_p (product inhibition) and K_i (inhibition by excess substrate) can be more challenging. In the first case, determination of the type of inhibition requires more complex experiments that are beyond the scope of the present contribution. In the second, the inhibition constant K_i can only be roughly estimated. In an experimental approach, we compared the results obtained either with initial rate measurements or with 50 to 60% conversion of the substrate. Similar values of V and K_m were obtained. Measurements involving the conversion of a large proportion of substrate are particularly advantageous when the assay method is difficult or time-consuming, or when obtaining the substrate presents experimental difficulties or involves substantial costs.