Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics

Koley, Aditya P.; Robinson, Richard; Friedman, Fred K.

doi:10.1016/s0300-9084(97)82528-x

Cited by 44 publications

(42 citation statements)

References 28 publications

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“…One possible mechanism of the ANF-mediated decrease in fluorescence of the BADAN and CPM probes is modulation by this effector of the partitioning between two conformers of the enzyme. This interpretation is consistent with the hypothesis of Koley and co-authors that the mechanism of action of ANF involves redistribution of the protein between two conformers with different substrate specificity and different accessibility of the heme pocket (21,25,38). Another possibility is that the binding of ANF quenches the fluorescence of the label in the fluorescent conformer by some local changes in the proximity of the label that increase the probability of PET from Trp-72 and/or Tyr-68 to the label without any changes in the partitioning of fluorescent and completely quenched conformers.…”

Section: Discussionsupporting

confidence: 93%

“…At the same time, our recent studies on CYP3A4 emphasize an important role of persistent conformational heterogeneity in the mechanisms of interactions with substrates and allosteric ligands (30,37). Recently we proposed that this heterogeneity, which was first reported by Koley et al (21,38,39), may originate from differences in conformation and/or orientation of the subunits in P450 oligomers (37). We also suggested that the mechanism of action of heterotropic activators, such as ANF, may result from modulation of the architecture of the oligomer, which changes the partitioning between different conformers of the enzyme (30,37).…”

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confidence: 92%

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Mechanism of Interactions of α-Naphthoflavone with Cytochrome P450 3A4 Explored with an Engineered Enzyme Bearing a Fluorescent Probe

et al. 2007

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Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into α-helix A‥ The site of modification was identified as Cys-64 with the help of CYP3A4 (C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN), 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and α-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of BADAN-modified enzyme is characterized by an S 50 of 18.2 ± 0.7, compared with the value of 2.2 ± 0.3 for the ANF-induced spin transition, thus revealing an additional low affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H 2 O 2 -dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer. KeywordsCytochrome P450 3A4; α-naphthoflavone; cooperativity; substrate binding site; BADAN; conformational heterogeneity A number of recent studies on the mechanisms of function and regulation of microsomal monooxygenase systems have focused on the importance of homo-and heterotropic cooperativity exhibited by various mammalian drug-metabolizing cytochromes P450 (1-8). Cooperative behavior has been reported for such enzymes as P450 1A2 (9), 2C9 (10-12), 2C5 (13), 2B6 (14), and 2B4 (15). However, the most prominent cases of P450 cooperativity are found in cytochrome P450 3A4 (CYP3A4), the principal drug-metabolizing enzyme in humans † This research was supported by NIH grant GM54995, Center grant ES06676, and research grant H-1458 from the Robert A. Welch NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 28. Published in final edited form as:Biochemistry. 2007 January 9; 46(1): 106-119. doi:10.1021/bi061944p. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (16). In addition to the homotropic cooperativity observed with a wide variety of substrates (1,4,(16)(17)(18), CYP3A4 also provides important examples of heterotropic activation (1...

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Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 92%

Mechanism of Interactions of α-Naphthoflavone with Cytochrome P450 3A4 Explored with an Engineered Enzyme Bearing a Fluorescent Probe

et al. 2007

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“…A possible explanation for these findings comes from the very complex behaviour of human CYP3A4. It appears that for this enzyme multiple conformers with distinct substrate specificities may exist (Koley et al 1996(Koley et al & 1997. Futhermore, cooperativity in oxidations catalysed by the enzyme was described and attributed to the existence of more than one binding site at the CYP3A4 molecule (Ueng et al 1997).…”

Section: Discussionmentioning

confidence: 99%

“…In the case of CYP3A4, two model substrates were used, i.e. denitronifedipine (Böcker et al 1986) and erythromycin (Riley & Howbrook 1997;Wang et al 1997), because for this enzyme multiple conformers with distinct substrate specificity may exist (Koley et al 1996(Koley et al & 1997.…”

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confidence: 99%

Inhibitory Effects of Silibinin on Cytochrome P‐450 Enzymes in Human Liver Microsomes

Beckmann-Knopp¹,

Rietbrock²,

Weyhenmeyer³

et al. 2000

Pharmacology & Toxicology

130

101

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Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7-300 mM) at 37ae in 0.1 M KH 2 PO 4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC 50 values were determined for each substrate at respective K M concentrations. Silibinin had little effect (IC 50 Ͼ200 mM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(π)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC 50 Ω173 mM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC 50 Ω29 mM and 46 mM) and S(ª)-warfarin 7-hydroxylation (CYP2C9; IC 50 Ω43 mM and 45 mM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (K i,c Ω2.3 mM and 2.4 mM). Inhibition was competitive for S(ª)-warfarin 7-hydroxylation (K i,c Ω18 mM and 19 mM) and mainly non-competitive for denitronifedipine oxidation (K i,n Ω9 mM and 12 mM). With therapeutic silibinin peak plasma concentrations of 0.6 mM and biliary concentrations up to 200 mM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.

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“…Elucidating the mechanisms of CYP3A4 cooperativity is complicated by the persistent conformational heterogeneity observed, whereby the enzyme is thought to be represented by at least two conformers with different ligand-binding properties [19][20][21][22][23][24]. This functional heterogeneity appears to be linked closely to the oligomeric state of the enzyme [19,20].…”

Section: Introductionmentioning

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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Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics

Cited by 44 publications

References 28 publications

Mechanism of Interactions of α-Naphthoflavone with Cytochrome P450 3A4 Explored with an Engineered Enzyme Bearing a Fluorescent Probe

Mechanism of Interactions of α-Naphthoflavone with Cytochrome P450 3A4 Explored with an Engineered Enzyme Bearing a Fluorescent Probe

Inhibitory Effects of Silibinin on Cytochrome P‐450 Enzymes in Human Liver Microsomes

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

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