title: Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity.
abstract: The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W, and L211F/D214E was studied with fluorescence resonance energy transfer and absorbance spectroscopy. Although in the wild type enzyme, the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (n(H)) around 1.7 (S(50) = 11.7 µM), the mutants showed no cooperativity (n(H) ≤ 1.1) with unchanged S(50) values. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high-affinity interaction is characterized by a dissociation constant (K(D)) ≤ 1.0 µM, whereas the K(D) of the second binding has the same magnitude as the S(50). Theoretical analysis of a two-step binding model suggests that n(H) values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event. Alteration of cooperativity in the mutants is caused by a partial displacement of the "spin-shifting" step. Although in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift on binding of the first substrate molecule.
authors.affiliation: Department of Pathology, The University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Harshica
authors.initials: H
authors.lastname: Fernando
authors.forename: Jessica A O
authors.initials: JA
authors.lastname: Rumfeldt
authors.forename: Nadezhda Y
authors.initials: NY
authors.lastname: Davydova
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
mesh_terms: D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites; D001971:Bromocriptine / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000235:genetics* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011721:Pyrenes / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C463480:1-pyrenebutanol; D050505:Mutant Proteins; D011721:Pyrenes; D001971:Bromocriptine; D009320:Atrial Natriuretic Factor; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human