Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes

Renaud, Jean‐Paul; Davydov, Dmitri R.; Heirwegh, K. P. M.; Mansuy, Daniel; Hoa, G H Hui Bon

doi:10.1042/bj3190675

Cited by 48 publications

(67 citation statements)

References 33 publications

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“…The analysis of the series of absorbance and fluorescence spectra was done with our SpectraLab program using a principal component analysis (PCA) method (also known as singular value decomposition (SVD) technique), as described earlier [12,54,55]. To interpret the changes in absorbance spectra in terms of the changes in the concentration of P450 species, we used a least-squares fitting of the spectra of principal components by the set of the spectral standards of pure high-and low-spin of CYP3A4 [37] complemented, when appropriate, with the extinction spectra of thiol-and thiolate complexes of P450cam taken from [56].…”

Section: Data Processingmentioning

confidence: 99%

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Davydov

Davydova

Tsalkova

et al. 2008

Archives of Biochemistry and Biophysics

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Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl) coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.

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Section: Data Processingmentioning

confidence: 99%

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Davydov

Davydova

Tsalkova

et al. 2008

Archives of Biochemistry and Biophysics

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“…The series of absorbance and fluorescence spectra obtained in titration experiments were analyzed using principal component analysis (PCA) as described previously [14,40,58,59]. 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].…”

Section: Data Processingmentioning

confidence: 99%

“…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].…”

Section: Data Processingmentioning

confidence: 99%

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

Fernando

Davydov

Chin

et al. 2007

Archives of Biochemistry and Biophysics

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The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S 50 = 5.4 μM, n = 1.0) but retained cooperativity of α-naphthoflavone binding. Continuous variation (Job's titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high affinity site (K D = 0.3 μM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6β-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo-and apo-P450 in mixed oligomers.

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“…The series of absorbance and fluorescence spectra obtained in titration experiments were analyzed using principal component analysis method (PCA) as described previously (39,40). This approach, which is illustrated in Supporting Information, allowed us to increase the signalto-noise ratio and improve the accuracy of the assay by sorting out the changes arising from the absorbance of the substrate and the changes in the light scattering during the experiment (17,22).…”

Section: Data Processingmentioning

confidence: 99%

“…This approach, which is illustrated in Supporting Information, allowed us to increase the signalto-noise ratio and improve the accuracy of the assay by sorting out the changes arising from the absorbance of the substrate and the changes in the light scattering during the experiment (17,22). To interpret the changes in the spectra of absorbance in terms of the concentration of P450 low-spin, high-spin and P420 species we used a least-squares fitting of the spectra of the first and second principal components to the set of spectral standards of the pure high spin, substrate-free ferric low-spin (16,39,40) and Type II-substrate-bound ferric low-spin CYP3A4. The latter spectral standard with the Soret band at 425 nm (ε = 114 mM −1 cm −1 ), α-band at 571 nm (ε = 10 mM −1 cm −1 ) and β-band at 538 nm (ε = 15 mM −1 cm −1 ) was obtained from the titration of CYP3A4 with imidazole.…”

Section: Data Processingmentioning

confidence: 99%

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

2006

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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...

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Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes

Cited by 48 publications

References 33 publications

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

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

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

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