Simplified approaches to kinetic isotope effects in cytochrome P450-catalyzed reactions and relevance to deuterated drugs

Kinetic isotope effects (KIEs) have been used to study mechanisms of cytochrome P450 (P450, CYP) reactions and are the basis of most applications for deuterated drugs. However, the complexity of KIEs has led to limited use and to some erroneous conclusions. Kinetic data for the 21-hydroxylation of the steroid 17α-hydroxyprogesterone by P450 21A2 (Pallan et al. 2015.J. Biol. Chem. 290:13128-13143) were utilized with modern kinetic analysis software (KinTek Explorer®) and simplified models.Reanalysis confirms the importance of several previous observations: (i) KIEs on the ratio kcat/Km (D(V/K), i.e. H(kcat/Km)/D(kcat/Km)) are most relevant; (ii) proper modeling of P450 reactions requires consideration of side reactions, both for substrate(s) oxidation and oxygen reduction, and (iii) a slow rate constant for a step(s) following product formation has a dramatic effect on D(V/K) due to perturbation of Km. Some applications of KIEs to the design and development of deuterated drugs are presented.