atrial-natriuretic-factor and 1-pyrenebutanol

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.

Topics: Atrial Natriuretic Factor; Binding Sites; Bromocriptine; Cytochrome P-450 CYP3A; Fluorescence Resonance Energy Transfer; Humans; Mutant Proteins; Protein Binding; Pyrenes; Substrate Specificity

P450eryF is the only bacterial P450 to show cooperativity of substrate binding and oxidation. However, the studies reported so far have provided evidence only for homotropic cooperativity of P450eryF but not for heterotropic cooperativity. Therefore, oxidation of 7-benzyloxyquinoline (7-BQ) and 1-pyrenebutanol (1-PB) by P450eryF A245T and spectral binding of 9-aminophenanthrene (9-AP) to wild-type P450eryF were investigated in the presence of various effectors. The addition of steroids and flavones caused no stimulation but rather moderate inhibition of 7-BQ or 1-PB oxidation by P450eryF A245T. However, the binding affinity of 9-AP was significantly increased in the presence of androstenedione or alpha-naphthoflavone (ANF). A comparative study with CYP3A4 revealed a similar increase in the binding affinity of 9-AP for the enzyme at low ANF concentrations but some competition at higher ANF concentrations. These studies, to our knowledge, provide the first report of heterotropic cooperativity in P450eryF as well as spectroscopic evidence for simultaneous presence of two ligand molecules in the CYP3A4 active site.

Topics: Alanine; Androstenedione; Atrial Natriuretic Factor; Bacteria; Bacterial Proteins; Binding Sites; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Flavones; Flavonoids; In Vitro Techniques; Mixed Function Oxygenases; Mutagenesis, Site-Directed; Oxidation-Reduction; Pyrenes; Quinolines; Structure-Activity Relationship; Substrate Specificity; Threonine; Titrimetry