Steady state kinetic model constraint for Multivariate Curve Resolution-Alternating Least Squares analysis

Enzyme incubations, when analyzed by liquid chromatography coupled to mass spectrometry (LC-MS), produce three-way data that are dependent on the initial substrate concentration(s), the mass-to-charge ratio (m/z) and the retention time. These data can be used to obtain kinetic parameters for the enzyme catalyzed reaction. In this paper, we present a new steady-state constraint for Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS). The derivation of the steady state rate equations is achieved assuming rapid equilibrium between the enzyme species involved in the mechanism. e of this new steady state kinetic constraint embedded into MCR-ALS makes it possible to fit three-way LC-MS enzyme incubation data to extract enzyme kinetic parameters such as the Michaelis constant (KM), the maximum velocity (vmax), and inhibition constants (KI). Specifically, we show that the use of this MCR-ALS algorithm allows for the analysis of data that can be difficult to handle using other means, including the occurrence of isobaric metabolites, chromatographic peak overlap, variable background contributions, and chromatographic peak distortions caused by chromatographic column overloading and by matrix effects. We have applied this approach to the study of the simultaneous incubation of two cytochrome P450 isoenzymes, CYP2D6 and CYP3A4, with dextromethorphan, an over-the-counter cough suppressant.