Short Communication A Theoretical Validation of the Substrate Depletion Approach to Determining Kinetic Parameters

The substrate depletion approach is an increasingly popular alternative to the traditional method (observing product formation) of determining the kinetic parameters (KM and Vmax) of an enzyme. Obach and Reed-Hagen (Drug Metab Dispos 30:831–837, 2002) used an empirical relationship between substrate depletion rate constants and initial substrate concentration to determine kinetic parameters for a number of cytochrome P450-catalyzed reactions. We present a proof that this relationship can be derived from the Michaelis-Menten equation and, therefore, that kinetic parameters obtained by the substrate depletion approach are equivalent and comparable to those obtained by the traditional product formation approach. Analysis of a simulated data set produced similar kinetic parameters regardless of which approach was used, confirming the theoretical result. Traditionally, the kinetic behavior of an enzyme is characterized by monitoring rates of product formation at various substrate concentrations. However, it is also possible to determine the kinetic parameters of an enzymatic reaction by monitoring depletion of substrate as a function of time, for use in situations where experimental considerations make it impossible or impractical to monitor product formation. Substrate depletion is increasingly widely used to characterize cytochrome P450 kinetics (Obach and Reed-Hagen, 2002; Isoherranen et al., 2004; Jones and Houston, 2004; Komura and Iwaki, 2005; Mohutsky et al., 2006). Obach and Reed-Hagen (2002) proposed the following empirical equation to obtain the Michaelis constant, KM: