Concurrent Cooperativity and Substrate Inhibitionin the Epoxidation of Carbamazepine by Cytochrome P450 3A4 ActiveSite Mutants Inspired by Molecular Dynamics Simulations

摘要

Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provideevidence that the substrate-binding pocket of CYP3A4 can accommodatemore than one molecule of CBZ. Analysis of the kinetics profiles ofCBZ metabolism with a model that combines the formalism of the Hillequation with an allowance for substrate inhibition demonstrates thatthe mechanism of interactions of CBZ with CYP3A4 involves multiplesubstrate-binding events (most likely three). Despite the retentionof the multisite binding mechanism in the mutants, functional manifestationsreveal an exquisite sensitivity to even minor structural changes inthe binding pocket that are introduced by conservative substitutionssuch as I369F, I369L, and A370V.