Autoactivation and activation of the cytochrome P450s

Objective: In order to reliably predict in vivo pharmacokinetic parameters from in vitro data, we must thoroughly understand the systems we currently use to determine enzyme kinetic parameters. There have been a number of rep orts of atypical Michaelis-Menten kinetics for cytochrome- (CYP) P4503A med iated metabolism in vitro but little discussion of its clinical relevance. In this manuscript, we examined the scope of CYP autoactivation and confirm ed that CYP1A2 demonstrates atypical Michaelis-Menten kinetics in vitro. Ma terials: Human liver microsomes, baculovirus-expressed CYP1A2, CYP1A2 in th e RECO format, and E. coli expressed CYP1A2 were utilized. Methods: Enzyme kinetics were performed using the various human CYP1A2 sources and ethoxyre sorufin O-deethylation as a prototypical biotransformation. The data were f it to various models of enzyme kinetics. In some cases the data best fit th e Hill equation, which was used to empirically model allosteric-type autoac tivation kinetics. Results: RECO CYP1A2 and E. coli expressed CYP1A2 both d emonstrated autoactivation kinetics for ethoxyresorufin O-deethylation. Whe n the data were fit to the Hill equation, n (the slope factor) was found to be 1.4 and 1.8 for RECO and E. coli expressed CYP1A2, respectively. Human Liver microsomal and insect expressed sources of CYP1A2 illustrated classic al Michaelis-Menten kinetics for the O-deethylation of ethoxyresorufin. Con clusion: Data generated in the current study and previous work suggest many CYPs, not only CYP3A, appear to behave as allosteric enzymes. We would arg ue that this is not necessarily a classical allosteric mechanism because n is frequently a non-integer. This autoactivation appears to be a function o f several factors including substrate physicochemical characteristics, spec ific interactions of the substrates (activators) with the enzyme active sit e, and presence of other enzyme modulators. These factors interact to incre ase the catalytic activity of CYP and thus the complexity of predicting enz yme kinetic parameters or drug interactions.