Resolution of Multiple Substrate Binding Sites in Cytochrome P450 3A4:  The Stoichiometry of the Enzyme−Substrate Complexes Probed by FRET and Job's Titration

Abstract

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the Type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 ± 0.3 (S 50 = 8.0 ± 1.1 µM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting K D values of 0.31 ± 0.08 µM and 6.7 ± 1.9 µM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with K D values of 0.16 ± 0.09, 4.8 ± 1.4, and 0.18 ± 0.09 µM for 1-PB, PMA, and bromocriptine respectively. Correspondingly, the Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of K D obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.Keywords substrate binding; cooperativity; conformers; spin equilibrium Recent studies of function and regulation of cytochromes P450 reveal increasing attention to the mechanisms and pharmacological significance of homo-and heterotropic cooperativity observed in various mammalian P450 species (1-8). The most prominent examples of these phenomena are observed with cytochrome P450 3A4 (CYP3A4), the most abundant P450 in human liver. CYP3A4 is responsible for the metabolism of a broad range of drug substrates, and studies of cooperativity of this enzyme are of vital importance to our efforts to elucidate the mechanisms of drug metabolism in humans (9). However, despite extensive studies, a general mechanism of cooperativity remains obscure. † This research was supported by NIH grants GM54995 (JRH), and Center grant ES06676 (JRH). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe prevailing hypothesis is that cytochromes P450 exhibiting cooperativity accommodate multiple substrate molecules in one large binding pocket (10)(11)(12). A loose fit of a single substrate molecule requires the binding of a second ligand for efficient binding and/or catalysis (11-13). However, the possibility that P450 cooperativity reflects a true case of a...

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

“…Here the excitation bandwidth was set to 10 nm. The spectra of emission were recorded in the 360 -570 nm range and corrected for the changes in the intensity of the excitation light during the experiment as described [48].…”

“…The variable path length continuous variation (Job's titration) experiments were carried out with a S2000 spectrometer (Ocean Optics Inc.) equipped with a custom-designed fiber optic adapter for a 10-cm long cylindrical cell (Cell Type-521, NSG Precision Cells, Farmingdale, NY) as described previously [48,49]. All experiments were carried out at 25 °C in 100 mM HEPES buffer (pH 7.4), containing 1 mM DTT and 1 mM EDTA.…”

“…To interpret the spectral transitions in terms of the concentration of P450 species we used a leastsquares fitting of the spectra of principal components to the set of spectral standards of pure low-spin, high-spin and P420 species of the hemeprotein [49,58,59]. The series of spectra obtained in continuous variation with variable optical path length were normalized to the path length prior to further analysis [48,49]. All data treatment procedures and curve fitting were performed using our SPECTRALAB software package [58].…”

“…Although most analysis of P450 cooperativity has been based on steady-state kinetics of substrate oxidation, more recent attention has focused on investigating the substrate binding step in the CYP3A4 catalytic cycle [20,[40][41][42][43][44][45][46][47][48][49]. In general, cooperativity in substrate binding is revealed by a sigmoidal curve of the substrate-induced displacement of the spin equilibrium of the heme iron, which is known to be an important determinant of the catalytic efficiency and coupling of cytochromes P450 [50][51][52].…”

“…Recent use of fluorescence resonance energy transfer (FRET) from the fluorescent substrate 1-pyrenebutanol (1-PB) to the heme of cytochrome P450eryF (P450 107A1) [14,15,40] or CYP3A4 [48] has allowed us to determine the dissociation constant of the first binding event directly. The results suggest that both enzymes possess two binding sites with different affinities for 1-PB.…”