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Research ArticleArticle

ROLE OF CYTOCHROME B5 IN MODULATING PEROXIDE-SUPPORTED CYP3A4 ACTIVITY: EVIDENCE FOR A CONFORMATIONAL TRANSITION AND CYTOCHROME P450 HETEROGENEITY

Santosh Kumar, Dmitri R. Davydov and James R. Halpert
Drug Metabolism and Disposition August 2005, 33 (8) 1131-1136; DOI: https://doi.org/10.1124/dmd.105.004606
Santosh Kumar
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Dmitri R. Davydov
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James R. Halpert
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Abstract

The role of cytochrome b5 (b5) in the α-naphthoflavone (α-NF)-mediated inhibition of H2O2-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although α-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H2O2 system. Analysis of the effect of various constituents of a standard reconstituted system on H2O2-supported activity showed that b5 alone resulted in a 2.5-fold increase in the kcat value and reversed the inhibitory effect of α-NF. In addition, titration with b5 suggested that only 65% of the CYP3A4 participated in the interaction with b5, consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b5 on the kinetics of H2O2-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b5, 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b5 decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b5 action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b5. Ser-119 is in the B′-C loop, a predicted b5-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b5 with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool.

Footnotes

  • This work was supported by National Institutes of Health Grant GM54995 and Center Grant ES06676.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.105.004606.

  • ABBREVIATIONS: α-NF, α-naphthoflavone; P450, cytochrome P450; 7-BQ, 7-benzyloxyquinoline; 7-BFC, 7-benzyloxy-4-(trifluoromethyl)coumarin; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; SRS, substrate recognition site; CPR, NADPH-cytochrome P450 reductase; b5, cytochrome b5; H2O2, hydrogen peroxide; CuOOH, cumene hydroperoxide; P450eryF, 6-deoxyerythronolide B hydroxylase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DOPC, dioleoylphosphatidylcholine.

    • Received March 2, 2005.
    • Accepted April 29, 2005.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 33 (8)
Drug Metabolism and Disposition
Vol. 33, Issue 8
1 Aug 2005
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Research ArticleArticle

ROLE OF CYTOCHROME B5 IN MODULATING PEROXIDE-SUPPORTED CYP3A4 ACTIVITY: EVIDENCE FOR A CONFORMATIONAL TRANSITION AND CYTOCHROME P450 HETEROGENEITY

Santosh Kumar, Dmitri R. Davydov and James R. Halpert
Drug Metabolism and Disposition August 1, 2005, 33 (8) 1131-1136; DOI: https://doi.org/10.1124/dmd.105.004606

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Research ArticleArticle

ROLE OF CYTOCHROME B5 IN MODULATING PEROXIDE-SUPPORTED CYP3A4 ACTIVITY: EVIDENCE FOR A CONFORMATIONAL TRANSITION AND CYTOCHROME P450 HETEROGENEITY

Santosh Kumar, Dmitri R. Davydov and James R. Halpert
Drug Metabolism and Disposition August 1, 2005, 33 (8) 1131-1136; DOI: https://doi.org/10.1124/dmd.105.004606
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