Theory for the observed isotope effects on the formation of multiple products by different kinetic mechanisms of cytochrome P450 enzymes

Abstract

Cytochrome P450 systems are unusual in that many of them can convert a substrate to a number of different metabolites. Several kinetic mechanisms may be envisioned by which the metabolites may be formed. In each of the mechanisms, the substrate combines with the enzyme in different orientations to form a set of (ES) complexes that then are activated to a set of (EOS) complexes. The fate of these (EOS) complexes determines the kinetic mechanism. In the "parallel pathway" mechanism, the (EOS) complexes are so stable and rigid they cannot be converted either directly or indirectly to complexes with different orientations; the orientation of the (ES) complexes thus determines which metabolite will be formed. In the "nondissociative" mechanisms, the complexes are not rigid; instead they undergo interconversion while the substrate remains in the active site of the enzyme. In the "dissociative" mechanisms, the (EOS) complexes dissociate to (EO) and (S), but recombine to form (EOS) complexes with either the same or different orientations. Steady-state equations describing the deuterium isotope effects for these kinetics mechanisms have been derived and solved for competitive experiments, in which equal concentrations of both deuterated and nondeuterated substrates are present in incubation mixtures, and for noncompetitive experiments, in which only one of the substrates is present. The equations reveal that comparisons of the isotope effects on the formation of a metabolite by a pathway that does not involve the abstraction of a deuterium from a deuterated substrate (the non-deuterium abstraction pathway) in both experiments can differentiate between the kinetic mechanisms. A value of 1.0 for (v)H/(v)D in the competitive experiment, but < 1.0 to > 1.0 for the value of (Vmax/Km)H/(Vmax/Km)D in the noncompetitive experiment, is diagnostic for the "dissociative" mechanisms. Values of 1.0 in both kinds of experiments are diagnostic for the "parallel pathway" mechanism. Values of < 1.0 in both types of experiments are diagnostic for the "nondissociative" mechanisms. The equations also predict possible unusual substrate-inhibitor interactions when the "dissociative" mechanism is operative.