Role of Cytochrome B5 in Modulating Peroxide-Supported Cyp3a4 Activity: Evidence for a Conformational Transition and Cytochrome P450 Heterogeneity

Kumar, Santosh; Davydov, Dmitri R.; Halpert, James R.

doi:10.1124/dmd.105.004606

Cited by 35 publications

(45 citation statements)

References 37 publications

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“…Consistent with our earlier observation (58), biexponential kinetics of heme destruction suggests persistent conformational heterogeneity of CYP3A4, such that the protein appears to be distributed between two populations with different accessibility of the heme moiety to H 2 O 2 . Furthermore, the striking difference between the time course of heme depletion and of the fluorescence increase (Fig.…”

Section: Discussionsupporting

confidence: 75%

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: 75%

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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“…The resulting decrease in oxidase activity would account for the greater change in NADPH consumption relative to product formation. This line of reasoning is also consistent with the observation that when sufficient levels of hydrogen peroxide, in the absence of NADPH, are present to support catalysis by shunting into the P450 catalytic cycle, cyt b5 can still stimulate metabolite formation (Kumar et al, 2005). At very high concentrations of cyt b5, competition between CPR and cyt b5 at a purported common site would result in a decrease in electron transfer from NADPH, as well as formation of products, including hydrogen peroxide and water.…”

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confidence: 73%

CYP2C9 Protein Interactions with Cytochrome b5: Effects on the Coupling of Catalysis

Locuson¹,

Wienkers²,

Jones³

et al. 2007

Drug Metabolism and Disposition

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ABSTRACT:The hemoprotein cytochrome b 5 (cyt b5) has been demonstrated to affect the kinetics of drug oxidation by the microsomal cytochromes P450 (P450s). However, the mechanisms through which cyt b5 exerts these effects are variable and P450 isoform-dependent. Whereas the effects of cyt b5 on the major drug-metabolizing enzymes CYP2D6, CYP2E1, and CYP3A4 are well studied, fewer studies conducted over limited ranges of cyt b5 concentrations have been performed on CYP2C9. In the present study with CYP2C9, cyt b5 exerted complex actions upon P450 oxidative reactions by affecting the rate of metabolite formation, the consumption of NADPH by cytochrome P450 reductase, and uncoupling of the reaction cycle to hydrogen peroxide and water. Cytochrome b 5 devoid of the heme moiety (apo-b5) exhibited effects similar to those of native cyt b5. All rates were highly dependent on the cyt b5 to CYP2C9 enzyme ratio, suggesting that the amount of cyt b5 present in an in vitro incubation is an important factor that can have an impact on the reliability of extrapolating in vitro generated data to predict the in vivo condition. The major effects of cyt b5 are hypothesized to result from a cyt b5-induced conformational change in CYP2C9 that results in an increased collision frequency between the iron-oxygen species (Cpd I) and the substrate, and a decrease in the oxidase activity. Together, these findings suggest that cyt b5 can alter multiple steps in the P450 catalytic cycle via complex interactions with P450 and P450 reductase.

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“…4). Because the presence of b 5 in the incubation mixture has been reported to enhance the catalytic activity of P450 enzymes (Schenkman and Jansson, 2003;Kumar et al, 2005), additional experiments with CYP3A5 were conducted using CYP3A5 ϩ OR supplemented with b 5 , as well as CYP3A5 containing coexpressed OR and P450 b 5 (CYP3A5 ϩ OR ϩ b 5 ). Measurable levels of M1 and M2 were detected for both CYP3A5 preparations containing b 5 .…”

Section: Resultsmentioning

confidence: 99%

Cytochrome P450 3A-Dependent Metabolism of a Potent and Selective γ-Aminobutyric AcidAα2/3 Receptor Agonist in Vitro: Involvement of Cytochrome P450 3A5 Displaying Biphasic Kinetics

Polsky-Fisher²,

Cui³

et al. 2007

Drug Metabolism and Disposition

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ABSTRACT:In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine (TPA023), a selective agonist of human ␥-aminobutyric acid A receptor ␣2 and ␣3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (>85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. EadieHofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K m (3.4 and 4.5 M, respectively) and a high K m (12.7 and 40.0 M, respectively) component. For both metabolites, the high K m component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4 > CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (>75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p < 0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 M. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.

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Role of Cytochrome B5 in Modulating Peroxide-Supported Cyp3a4 Activity: Evidence for a Conformational Transition and Cytochrome P450 Heterogeneity

Cited by 35 publications

References 37 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

CYP2C9 Protein Interactions with Cytochrome b5: Effects on the Coupling of Catalysis

Cytochrome P450 3A-Dependent Metabolism of a Potent and Selective γ-Aminobutyric AcidAα2/3 Receptor Agonist in Vitro: Involvement of Cytochrome P450 3A5 Displaying Biphasic Kinetics

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