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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Dual Role of Human Cytochrome P450 3A4 Residue Phe-304 in Substrate Specificity and Cooperativity

Tammy L. Domanski, You-Ai He, Greg R. Harlow and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics May 2000, 293 (2) 585-591;
Tammy L. Domanski
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You-Ai He
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Greg R. Harlow
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James R. Halpert
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Abstract

The structural basis of cooperativity of progesterone hydroxylation catalyzed by human cytochrome P450 3A4 has been investigated. A recent study suggested that substitution of larger side chains at positions Leu-211 and Asp-214 partially mimics the action of effector by reducing the size of the active site. Based on predictions from molecular modeling that Phe-304 in the highly conserved I helix is involved in both effector and substrate binding, a tryptophan residue was substituted at this position. The purified F304W mutant displayed hyperbolic progesterone hydroxylase kinetics, indicating a lack of homotropic cooperativity. However, the mutant remained responsive to stimulation by α-naphthoflavone, exhibiting a 2-fold decrease in theKm value for progesterone 6β-hydroxylation in the presence of 25 μM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained theVmax and decreased theKm for progesterone 6β-hydroxylation, minimized stimulation by α-naphthoflavone, and decreased the rate of α-naphthoflavone oxidation to one-eighth of the wild type. Interestingly, the ΔAmax for spectral binding of α-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and α-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen.

Footnotes

  • Send reprint requests to: Dr. Tammy L. Domanski, Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031. E-mail: tadomans{at}utmb.edu

  • ↵1 This study was supported by National Research Award GM19058, National Institutes of Health Grant GM54995, and Center Grant ES06676.

  • Received for publication

  • Abbreviations:
    P450
    cytochrome P450
    ANF
    α-naphthoflavone
    PCR
    polymerase chain reaction
    DOPC
    dioloeoylphosphatidylcholine
    CHAPS
    3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
    E
    enzyme
    S
    substrate
    WT
    wild type
    • Accepted December 23, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 293 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 293, Issue 2
1 May 2000
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Dual Role of Human Cytochrome P450 3A4 Residue Phe-304 in Substrate Specificity and Cooperativity

Tammy L. Domanski, You-Ai He, Greg R. Harlow and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics May 1, 2000, 293 (2) 585-591;

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Dual Role of Human Cytochrome P450 3A4 Residue Phe-304 in Substrate Specificity and Cooperativity

Tammy L. Domanski, You-Ai He, Greg R. Harlow and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics May 1, 2000, 293 (2) 585-591;
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