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 continuous 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 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, 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 KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, 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 KD 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.

Figure 1

Interactions of CYP3A4 with 1-PB monitored by the substrate-induced spin shift. (a) A series of the absorbance spectra obtained at no substrate present and at 0.78, 1.6, 3.1, 5.5, 7.8, 10.1, 11.7, 17.2, 25.7, and 38.0 µM 1-PB. The insert shows the differential spectra obtained by subtraction of the first spectrum of the series (at no substrate present). (b) The same data shown as the plot of the percent of the high spin P450 versus the concentration of the substrate.. The line shows the approximation of this data set by the Hill equation with S₅₀ = 8.6 µM, n = 1.8, and the maximal amplitude of the spin shift of 48%. The reaction mixture contained 1.45 µM CYP3A4 in 0.1 M Na-Hepes buffer, pH 7.4, 1 mM DTT, 1 mM EDTA and was kept at 25 °C.

Figure 2

Interactions of CYP3A4 with PMA monitored by the substrate-induced Type-II spectral transition. (a) A series of the absorbance spectra obtained at no substrate present and at 0.42, 1.3, 1.7, 4.2 10.4, 12.4, 24.7, 30.8, and 48.9 µM PMA. The insert shows the differential spectra obtained by subtraction of the first spectrum of the series (at no substrate present). (b) The same data shown as the fraction of the (water-ligated) low spin state P450 versus the concentration of the substrate. The solid line shows the approximation of this data set with a binary association equation with K_D = 7.6 µM and the maximal amplitude of the changes in the content of the P450 low-spin state of 58%. The concentration of the enzyme was 1.2 µM. Other conditions as indicated in Fig. 1.

Figure 3

Changes in specific fluorescence of 1-PB (a), PMA (b), and bromocriptine (c) upon dilution of the mixture of CYP3A4 with the respective substrates at a 1:1 molar ratio. The signal was normalized based on protein concentration and corrected for the internal filter effect as described. The left panels represent the series of fluorescence spectra measured at 0.26, 0.53, 1.0, 1.5, 2.0, 3.9, 4.7, 5.5, 6.3, 7.0, and 14.0 µM (panel a); 0.33, 0.64, 1.0, 1.4, 2.0, 2.7, 3.7, 4.8, 6.6, 8.1, and 8.9 µM (panel b); and 1.9, 1.6, 1.4, 1.1 and 0.83 µM enzyme (panel c). In all cases the increase in the concentration of enzyme/substrate mixture caused a decrease in the normalized intensity of emission. The right panels represent the titration curves obtained from these data sets using principal component analysis, as described. The solid lines represent the approximations of the data sets by the equation for the equilibrium of bimolecular association with the values of K_D1 of 0.25 µM, 3.1 µM, and 0.20 µM for 1-PB, PMA, and bromocriptine respectively. The insets show the spectra of the first (solid lines) and the second (dashed lines) principal components scaled according to the maximal changes in the respective component observed in these titrations. Conditions as indicated in Fig 1.

Figure 4

Titration-by-dilution experiment on the interactions of CYP3A4 with 1-PB detected by absorbance spectroscopy at excess substrate. (a) A series of absorbance spectra of 1-PB in mixture with P450 3A4 at a 1:11 molar ratio. The spectra shown were obtained at 16.6 (initial concentration), 8.8, 5.3, 3.8, 3.3, 2.0, 1.5, 1.1, 0.83, and 0.66 µM of CYP3A4. The inset shows a series of difference spectra of Fig. 4A. The experiment was carried out in a 10-cm vertical cell with the optical pathlength increasing with the dilution of the sample. The amplitudes of the spectra were corrected for non-linearity of the dependence of the path length on the volume of the sample. (b) The spectral change shown in Fig. 4A as the plot of the fraction high spin P450 3A4 versus the concentration of the heme protein. The solid line shows the approximation of this data set with a binary association equation with a K_D2 = 8.5 µM and the maximal amplitude of the spin transition of 54%. Conditions as indicated in Fig. 1.

Figure 5

Titration-by-dilution experiment on the interactions of CYP3A4 with PMA detected by absorbance spectroscopy at excess substrate. (a) A series of absorbance spectra of PMA in mixture with P450 3A4 at 1:13 molar ratio in a dilution setup. The spectra shown were obtained 32 at 7.9 (initial concentration), 4.0, 1.5, 1.1, 0.85, 0.66, 0.54, 0.49, 0.37, and 0.23 µM of CYP3A4. The inset shows a series of difference spectra of Fig. 5A. The experiment was carried out in a 10-cm vertical cell with the optical pathlength increasing with the dilution of the sample. The amplitudes of the spectra were corrected for non-linearity of the dependence of the path length on the volume of the sample. (b) The spectral change shown in Fig. 5A as the plot of the fraction of low spin P450 versus the concentration of the heme protein. The solid line shows the approximation of this data set with a binary association equation with a K_D1 = 3.6 µM and the maximal amplitude of the changes in the content of the P450 low-spin state of 82%. Conditions as indicated in Fig. 1.

Figure 6

Job’s titration of CYP3A4 with bromocriptine (a), PMA (b), and 1-PB (c). The experiments were carried out at 25 °C in a 10-cm vertical cell with the optical pathlength increasing with the dilution of the sample. CYP3A4 (500 µL) in 0.1 M Na-Hepes buffer, pH 7.4, 1 mM DTT, 1 mM EDTA was placed into the optical cell, and the substrate solution of the same concentration as the initial protein was added gradually, until a volume of 5.1 mL, when the cell was completely filled. The sum of the concentration of the enzyme and the substrate was therefore kept constant during the experiment, and was equal to 3 µM in the titration with bromocriptine and 12 µM in the experiments with 1-PB and PMA. The solid lines in panels (a) and (b) represent the fitting of the experimental data with the bimolecular association isotherm (Eq. 1) with the values of dissociation constants of 0.38 µM and 11.0 µM for bromocriptine and PMA, respectively. The dashed line in panel (c) represents the approximation of the data with the parallel binding model (Eq. 3), while the solid line shows their approximation with the equation derived for the sequential binding mechanism (Eq. 4), assuming the value of K_D1 equal to 0.16 µM, as determined in our FRET exoperiments. The respective values of the dissociation constants of the ternary enzyme-substrate complex (K_D2) are equal to 0.55 µM and 4.0 µM.