Conditions that can lead to biased kinetic parameters in reactions with enzymes simultaneously inhibited by reaction product and another inhibitor

In reactions where the enzyme is inhibited by the product, using the classical method based on initial rates to determine kinetic parameters may give rise to biased values. In fact, under certain conditions, failure to include the effect of product inhibition may generate errors of such magnitude that estimated parameters may not deserve any credit. In this work, three substrate depletion thresholds were used to obtain initial rates in reactions inhibited by the product and by a second different inhibitor. Kinetic parameters were determined by Michaelis–Menten rate equation using initial rates obtained from the slopes of theoretical curves product versus time simulated by MATLAB (in silico data). Errors in estimated parameters, including those of second inhibitor (K ic2 and K iu2 ), were quantified for each of the three substrate thresholds. As expected, in the absence of reaction product inhibition, i.e., when its inhibition constant tends towards infinity, minimal errors are observed in remaining estimated parameters. However, depending on the magnitude of product inhibition, percentage error in estimated parameter values can exceed 100%, being influenced by the parameter type (K ic2 and K iu2 are the most biased) and also by the substrate depletion threshold for determining initial rates (greater bias when substrate depletion exceeds 1%). In this work, we prove that when product inhibition occurs, the kinetic constants determined from the initial rates are generally subject to errors and, therefore, this is not a reliable method. Thus, in order to avoid determining potentially biased kinetic parameters values, we suggest an alternative methodology.