title: Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.
abstract: 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 provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.
authors.affiliation: Department of Obstetrics, University Hospital Zurich , Zürich, Switzerland.
authors.forename: Christian S
authors.initials: CS
authors.lastname: Müller
authors.forename: Tim
authors.initials: T
authors.lastname: Knehans
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Bounds
authors.forename: Ursula
authors.initials: U
authors.lastname: von Mandach
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.forename: Amedeo
authors.initials: A
authors.lastname: Caflisch
authors.forename: Willem H
authors.initials: WH
authors.lastname: Koppenol
mesh_terms: D002220:Carbamazepine / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D004852:Epoxy Compounds; D050505:Mutant Proteins; D002220:Carbamazepine; D006418:Heme; D051544:Cytochrome P-450 CYP3A