HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harlow, G. R. Articles by Halpert, J. R. Search for Related Content PubMed PubMed Citation Articles by Harlow, G. R. Articles by Halpert, J. R. Social Bookmarking                      What's this?

Volume 272, Number 9, Issue of February 28, 1997 pp. 5396-5402
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Alanine-scanning Mutagenesis of a Putative Substrate Recognition Site in Human Cytochrome P450 3A4
ROLE OF RESIDUES 210 AND 211 IN FLAVONOID ACTIVATION AND SUBSTRATE SPECIFICITY

(Received for publication, October 22, 1996, and in revised form, December 23, 1996)

From the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721

Alanine-scanning mutagenesis was performed on amino acid residues 210-216 of cytochrome P450 3A4, the major drug-metabolizing enzyme of human liver. Mutagenesis of this region, which has been proposed to align with the C-terminal ends of F-helices from cytochromes P450_BM-3, P450_terp, and P450_cam, served as a test of the applicability of the substrate recognition site model of Gotoh (Gotoh, O. (1992) J. Biol. Chem. 267, 83-90) to P450 3A4. The results, using two steroid substrates, indicated that substitution of Ala for Leu²¹⁰ altered the responsiveness to the effector -naphthoflavone and the regioselectivity of testosterone hydroxylation. Replacement of Leu²¹¹ by Ala also decreased the stimulation by -naphthoflavone, whereas mutations at residues 212-216 had little effect. The diminished flavonoid responses of the 210 and 211 mutants were observed over a wide range of progesterone and -naphthoflavone concentrations. Further characterization was performed with the additional effectors -naphthoflavone, flavone, and 4-chromanone. The finding that P450 3A4 with one altered residue, Leu²¹⁰  Ala, can have both an altered testosterone hydroxylation profile and response to flavonoid stimulation provides evidence that the substrate binding and effector sites are at least partially overlapping.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Y. Kapelyukh, M. J. I. Paine, J.-D. Marechal, M. J. Sutcliffe, C. R. Wolf, and G. C. K. Roberts Multiple Substrate Binding by Cytochrome P450 3A4: Estimation of the Number of Bound Substrate Molecules Drug Metab. Dispos., October 1, 2008; 36(10): 2136 - 2144. [Abstract] [Full Text] [PDF]

				S. Kumar, Y. Zhao, L. Sun, S. S. Negi, J. R. Halpert, and B. K. Muralidhara Rational Engineering of Human Cytochrome P450 2B6 for Enhanced Expression and Stability: Importance of a Leu264->Phe Substitution Mol. Pharmacol., November 1, 2007; 72(5): 1191 - 1199. [Abstract] [Full Text] [PDF]

				I. G. Denisov, B. J. Baas, Y. V. Grinkova, and S. G. Sligar Cooperativity in Cytochrome P450 3A4: LINKAGES IN SUBSTRATE BINDING, SPIN STATE, UNCOUPLING, AND PRODUCT FORMATION J. Biol. Chem., March 9, 2007; 282(10): 7066 - 7076. [Abstract] [Full Text] [PDF]

				S.-J. Lee, I. P. van der Heiden, J. A. Goldstein, and R. H. N. van Schaik A New CYP3A5 Variant, CYP3A5*11, Is Shown to Be Defective in Nifedipine Metabolism in a Recombinant cDNA Expression System Drug Metab. Dispos., January 1, 2007; 35(1): 67 - 71. [Abstract] [Full Text] [PDF]

				S. Kumar, H. Liu, and J. R. Halpert Engineering of Cytochrome P450 3A4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis Drug Metab. Dispos., December 1, 2006; 34(12): 1958 - 1965. [Abstract] [Full Text] [PDF]

				E. M. Isin and F. P. Guengerich Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4 J. Biol. Chem., April 7, 2006; 281(14): 9127 - 9136. [Abstract] [Full Text] [PDF]

				J.-D. Marechal, J. Yu, S. Brown, I. Kapelioukh, E. M. Rankin, C. R. Wolf, G. C. K. Roberts, Mark. J. I. Paine, and M. J. Sutcliffe IN SILICO AND IN VITRO SCREENING FOR INHIBITION OF CYTOCHROME P450 CYP3A4 BY COMEDICATIONS COMMONLY USED BY PATIENTS WITH CANCER Drug Metab. Dispos., April 1, 2006; 34(4): 534 - 538. [Abstract] [Full Text] [PDF]

				K.-i. Fujita, Y. Ando, M. Narabayashi, T. Miya, F. Nagashima, W. Yamamoto, K. Kodama, K. Araki, H. Endo, and Y. Sasaki GEFITINIB (IRESSA) INHIBITS THE CYP3A4-MEDIATED FORMATION OF 7-ETHYL-10-(4-AMINO-1-PIPERIDINO)CARBONYLOXYCAMPTOTHECIN BUT ACTIVATES THAT OF 7-ETHYL-10-[4-N-(5-AMINOPENTANOIC ACID)-1-PIPERIDINO]CARBONYLOXYCAMPTOTHECIN FROM IRINOTECAN Drug Metab. Dispos., December 1, 2005; 33(12): 1785 - 1790. [Abstract] [Full Text] [PDF]

				S. Kumar, D. R. Davydov, and J. R. Halpert ROLE OF CYTOCHROME B5 IN MODULATING PEROXIDE-SUPPORTED CYP3A4 ACTIVITY: EVIDENCE FOR A CONFORMATIONAL TRANSITION AND CYTOCHROME P450 HETEROGENEITY Drug Metab. Dispos., August 1, 2005; 33(8): 1131 - 1136. [Abstract] [Full Text] [PDF]

				Z. Wen, J. Baudry, M. R. Berenbaum, and M. A. Schuler Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel Protein Eng. Des. Sel., April 1, 2005; 18(4): 191 - 199. [Abstract] [Full Text] [PDF]

				R. P. Gupta, Y. A. He, K. S. Patrick, J. R. Halpert, and N. H. Bell CYP3A4 Is a Vitamin D-24- and 25-Hydroxylase: Analysis of Structure Function by Site-Directed Mutagenesis J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1210 - 1219. [Abstract] [Full Text] [PDF]

				J. K. Yano, M. R. Wester, G. A. Schoch, K. J. Griffin, C. D. Stout, and E. F. Johnson The Structure of Human Microsomal Cytochrome P450 3A4 Determined by X-ray Crystallography to 2.05-A Resolution J. Biol. Chem., September 10, 2004; 279(37): 38091 - 38094. [Abstract] [Full Text] [PDF]

				P. A. Williams, J. Cosme, D. M. Vinkovic, A. Ward, H. C. Angove, P. J. Day, C. Vonrhein, I. J. Tickle, and H. Jhoti Crystal Structures of Human Cytochrome P450 3A4 Bound to Metyrapone and Progesterone Science, July 30, 2004; 305(5684): 683 - 686. [Abstract] [Full Text] [PDF]

				C.-S. Chen, J. T. Lin, K. A. Goss, Y.-a. He, J. R. Halpert, and D. J. Waxman Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics Mol. Pharmacol., May 1, 2004; 65(5): 1278 - 1285. [Abstract] [Full Text]

				Y. Yamaguchi, K. K. Khan, Y. A. He, Y. Q. He, and J. R. Halpert TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROME B5 AND OF SITE-DIRECTED MUTAGENESIS Drug Metab. Dispos., January 1, 2004; 32(1): 155 - 161. [Abstract] [Full Text] [PDF]

				J. Baudry, W. Li, L. Pan, M. R. Berenbaum, and M. A. Schuler Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome P450 monooxygenase Protein Eng. Des. Sel., August 1, 2003; 16(8): 577 - 587. [Abstract] [Full Text] [PDF]

				K. K. Khan, H. Liu, and J. R. Halpert Homotropic Versus Heterotopic Cooperativity of Cytochrome P450eryF: A Substrate Oxidation and Spectral Titration Study Drug Metab. Dispos., April 1, 2003; 31(4): 356 - 359. [Abstract] [Full Text] [PDF]

				J. Liu, S. S. Ericksen, D. Besspiata, C. W. Fisher, and G. D. Szklarz Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382 Drug Metab. Dispos., April 1, 2003; 31(4): 412 - 420. [Abstract] [Full Text] [PDF]

				K. K. Khan, Y. Q. He, M. A. Correia, and J. R. Halpert Differential Oxidation of Mifepristone by Cytochromes P450 3A4 and 3A5: Selective Inactivation of P450 3A4 Drug Metab. Dispos., September 1, 2002; 30(9): 985 - 990. [Abstract] [Full Text] [PDF]

				S. M. Fowler, J. M. Taylor, T. Friedberg, C. R. Wolf, and R. J. Riley CYP3A4 Active Site Volume Modification by Mutagenesis of Leucine 211 Drug Metab. Dispos., April 1, 2002; 30(4): 452 - 456. [Abstract] [Full Text] [PDF]

				K. K. Khan, Y. Q. He, T. L. Domanski, and J. R. Halpert Midazolam Oxidation by Cytochrome P450 3A4 and Active-Site Mutants: an Evaluation of Multiple Binding Sites and of the Metabolic Pathway That Leads to Enzyme Inactivation Mol. Pharmacol., March 1, 2002; 61(3): 495 - 506. [Abstract] [Full Text] [PDF]

				M. Spatzenegger, Q. Wang, Y. Q. He, M. R. Wester, E. F. Johnson, and J. R. Halpert Amino Acid Residues Critical for Differential Inhibition of CYP2B4, CYP2B5, and CYP2B1 by Phenylimidazoles Mol. Pharmacol., March 1, 2001; 59(3): 475 - 484. [Abstract] [Full Text]

				T. L. Domanski, C. Finta, J. R. Halpert, and P. G. Zaphiropoulos cDNA Cloning and Initial Characterization of CYP3A43, a Novel Human Cytochrome P450 Mol. Pharmacol., February 1, 2001; 59(2): 386 - 392. [Abstract] [Full Text]

				T. L. Domanski, Y.-A. He, G. R. Harlow, and J. R. Halpert Dual Role of Human Cytochrome P450 3A4 Residue Phe-304 in Substrate Specificity and Cooperativity J. Pharmacol. Exp. Ther., May 1, 2000; 293(2): 585 - 591. [Abstract] [Full Text]

				S. Ekins, G. Bravi, J. H. Wikel, and S. A. Wrighton Three-Dimensional-Quantitative Structure Activity Relationship Analysis of Cytochrome P-450 3A4 Substrates J. Pharmacol. Exp. Ther., October 1, 1999; 291(1): 424 - 433. [Abstract] [Full Text]

				J. C. Stevens, T. L. Domanski, G. R. Harlow, R. B. White, E. Orton, and J. R. Halpert Use of the Steroid Derivative RPR 106541 in Combination with Site-Directed Mutagenesis for Enhanced Cytochrome P-450 3A4 Structure/Function Analysis J. Pharmacol. Exp. Ther., August 1, 1999; 290(2): 594 - 602. [Abstract] [Full Text]

				S. M. Strobel, G. D. Szklarz, Y. q. He, M. Foroozesh, W. L. Alworth, E. S. Roberts, P. F. Hollenberg, and J. R. Halpert Identification of Selective Mechanism-Based Inactivators of Cytochromes P-450 2B4 and 2B5, and Determination of the Molecular Basis for Differential Susceptibility J. Pharmacol. Exp. Ther., July 1, 1999; 290(1): 445 - 451. [Abstract] [Full Text]

				D. J. Fraser, Y. Q. He, G. R. Harlow, and J. R. Halpert Use of Chimeric Enzymes and Site-Directed Mutagenesis for Identification of Three Key Residues Responsible for Differences in Steroid Hydroxylation between Canine Cytochromes P-450 3A12 and 3A26 Mol. Pharmacol., February 1, 1999; 55(2): 241 - 247. [Abstract] [Full Text]

				G. D. Szklarz and J. R. Halpert Molecular Basis of P450 Inhibition and Activation. Implications for Drug Development and Drug Therapy Drug Metab. Dispos., December 1, 1998; 26(12): 1179 - 1184. [Abstract] [Full Text]

				J. R. Halpert, T. L. Domanski, O. Adali, C. P. Biagini, J. Cosme, E. A. Dierks, E. F. Johnson, J. P. Jones, P. O. de Montellano, R. M. Philpot, et al. Structure-Function of Cytochromes P450 and Flavin-Containing Monooxygenases. Implications for Drug Metabolism Drug Metab. Dispos., December 1, 1998; 26(12): 1223 - 1231. [Abstract] [Full Text]

				Y. Kobayashi, S. M. Strobel, N. E. Hopkins, W. L. Alworth, and J. R. Halpert Catalytic Properties of an Expressed Cytochrome P450 2b1 From a Wistar-Kyoto Rat Liver cdna Library Drug Metab. Dispos., October 1, 1998; 26(10): 1026 - 1030. [Abstract] [Full Text]

				G. R. Harlow and J. R. Halpert Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics PNAS, June 9, 1998; 95(12): 6636 - 6641. [Abstract] [Full Text] [PDF]

				D. J. Fraser, R. Feyereisen, G. R. Harlow, and J. R. Halpert Isolation, Heterologous Expression and Functional Characterization of a Novel Cytochrome P450 3A Enzyme from a Canine Liver cDNA Library J. Pharmacol. Exp. Ther., December 1, 1997; 283(3): 1425 - 1432. [Abstract] [Full Text]

				J. Cupp-Vickery, R. Anderson, and Z. Hatziris Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity PNAS, March 28, 2000; 97(7): 3050 - 3055. [Abstract] [Full Text] [PDF]