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Research ArticleArticle

Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Jianguo Liu, Spencer S. Ericksen, Dan Besspiata, Charles W. Fisher and Grazyna D. Szklarz
Drug Metabolism and Disposition April 2003, 31 (4) 412-420; DOI: https://doi.org/10.1124/dmd.31.4.412
Jianguo Liu
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Spencer S. Ericksen
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Dan Besspiata
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Charles W. Fisher
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Grazyna D. Szklarz
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Abstract

Key residue Val-382 in P450 1A1 has been predicted to interact with the alkoxy chain of resorufin derivatives. Therefore, we undertook a detailed analysis of substrate mobility in the active site of the P450 1A1 homology model and assessed the effect of mutations at position 382. Dynamic trajectories of 7-methoxy-, 7-ethoxy-, and 7-pentoxyresorufin indicated that 7-ethoxyresorufin would be oxidized most efficiently by the wild-type enzyme. The Val-382→Ala mutation would increase the O-dealkylation of 7-pentoxyresorufin but decrease the oxidation of other substrates. In the case of the V382L mutant, the large bulk of Leu would block alkoxyresorufins from productive binding orientations leading to lowered activities. Binding free energy calculations for three substrates with 1A1 WT and two mutants indicated that binding constants would be similar for all enzyme-substrate combinations. Modeling predictions were tested experimentally. The plasmid containing the cDNA for human P450 1A1 modified for bacterial expression was altered to include a C-terminal PCR-generated six histidine domain to facilitate enzyme purification. The V382A and V382L mutants were constructed by site-directed mutagenesis and Escherichia coli–expressed enzymes purified using Ni-NTA affinity chromatography. The activity of the WT 1A1 was highest toward 7-ethoxyresorufin and lowest toward 7-pentoxyresorufin. Both mutants displayed a decrease inVmax with 7-methoxy- and 7-ethoxyresorufin, whereas for the V382A mutant, Vmax with 7-pentoxyresorufin was increased. No significant changes inKm were observed relative to the wild-type enzyme. The experimental results are thus in good agreement with modeling predictions.

Footnotes

  • ↵1 Current address: CEDRA Corporation, 8609 Cross Park Drive, Austin, TX 78754.

  • This work was supported by National Institutes of Health Grants CA85542 and RR16440 (G.D.S.) and by ES07628 (C.W.F.). Modeling studies were performed at the Computational Chemistry and Molecular Modeling Laboratory, Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV.

  • Abbreviations used are::
    P450
    cytochrome P450
    WT
    wild-type
    IPTG
    isopropyl-β-d-thiogalactopyranoside
    ALA
    δ-aminolevulinic acid
    CHAPS
    3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
    DLPC
    dilauroyl-l-3-phosphatidyl choline
    PMSF
    phenylmethanesulfonyl fluoride
    bp
    base pairs
    PAGE
    polyacrylamide gel electrophoresis
    DMSO
    dimethylsulfoxide
    • Received September 28, 2002.
    • Accepted December 20, 2002.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 31 (4)
Drug Metabolism and Disposition
Vol. 31, Issue 4
1 Apr 2003
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Research ArticleArticle

Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Jianguo Liu, Spencer S. Ericksen, Dan Besspiata, Charles W. Fisher and Grazyna D. Szklarz
Drug Metabolism and Disposition April 1, 2003, 31 (4) 412-420; DOI: https://doi.org/10.1124/dmd.31.4.412

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Research ArticleArticle

Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Jianguo Liu, Spencer S. Ericksen, Dan Besspiata, Charles W. Fisher and Grazyna D. Szklarz
Drug Metabolism and Disposition April 1, 2003, 31 (4) 412-420; DOI: https://doi.org/10.1124/dmd.31.4.412
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