Cooperative Binding of Acetaminophen and Caffeine within the P450 3A4 Active Site

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Abstract

Acetaminophen (N-acetyl-p-aminophenol, APAP) is a commonly used analgesic/antipyretic. When oxidized by P450, a toxic APAP metabolite is generated. Human P450 3A4 was expressed in Escherichia coli, purified, and reconstituted using artificial liposomes. Oxidation of APAP by P450 3A4, as detected by the formation of its glutathione adduct, was found to exhibit negative homotropic cooperativity with a Hill coefficient of 0.7. In the presence of caffeine, the observed kinetics were close to classical Michaelis–Menten kinetics with a Hill coefficient approaching 1. In order to probe for a potential repositioning of APAP within the P450 3A4 pocket in the presence of caffeine, NMR T₁ paramagnetic relaxation techniques were used to calculate distances from the P450 3A4 heme iron to protons of APAP alone and in the presence of caffeine. Both APAP and caffeine were found to bind at the active site in proximity to the heme iron. When APAP was incubated with P450 3A4, the acetamido group of APAP was found to be closest to the heme iron consistent with the amide group of APAP weakly associating with the heme iron. The addition of caffeine disrupted the ability of APAP to coordinate with the heme iron of P450 3A4 and enhanced the rate of oxidation to its toxic metabolite.

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This article has been cited by 3 ACS Journal articles (3 most recent appear below).

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  The Structural Basis for Homotropic and Heterotropic Cooperativity of Midazolam Metabolism by Human Cytochrome P450 3A4

  Arthur G. Roberts, Jing Yang, James R. Halpert, Sidney D. Nelson, Kenneth T. Thummel, and William M. Atkins
  Biochemistry2011 50 (50), 10804-10818

  • The Structural Basis for Homotropic and Heterotropic Cooperativity of Midazolam Metabolism by Human Cytochrome P450 3A4

    Arthur G. Roberts, Jing Yang, James R. Halpert, Sidney D. Nelson, Kenneth T. Thummel, and William M. Atkins
    Biochemistry2011 50 (50), 10804-10818

    Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the ...

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• 

  NMR-Derived Models of Amidopyrine and Its Metabolites in Complexes with Rabbit Cytochrome P450 2B4 Reveal a Structural Mechanism of Sequential N-Dealkylation

  Arthur G. Roberts, Sara E. A. Sjögren, Nadezda Fomina, Kathy T. Vu, Adah Almutairi, and James R. Halpert
  Biochemistry2011 50 (12), 2123-2134

  • NMR-Derived Models of Amidopyrine and Its Metabolites in Complexes with Rabbit Cytochrome P450 2B4 Reveal a Structural Mechanism of Sequential N-Dealkylation

    Arthur G. Roberts, Sara E. A. Sjögren, Nadezda Fomina, Kathy T. Vu, Adah Almutairi, and James R. Halpert
    Biochemistry2011 50 (12), 2123-2134

    To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray ...

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• 

  Structures of Cytochrome P450 2B4 Complexed with the Antiplatelet Drugs Ticlopidine and Clopidogrel,

  Sean C. Gay, Arthur G. Roberts, Keiko Maekawa, Jyothi C. Talakad, Wen-Xu Hong, Qinghai Zhang, C. David Stout, and James R. Halpert
  Biochemistry2010 49 (40), 8709-8720

  • Structures of Cytochrome P450 2B4 Complexed with the Antiplatelet Drugs Ticlopidine and Clopidogrel,

    Sean C. Gay, Arthur G. Roberts, Keiko Maekawa, Jyothi C. Talakad, Wen-Xu Hong, Qinghai Zhang, C. David Stout, and James R. Halpert
    Biochemistry2010 49 (40), 8709-8720

    Prior X-ray crystal structures of rabbit cytochrome P450 2B4 (2B4) in complexes with various imidazoles have demonstrated markedly different enzyme conformations depending on the size of the inhibitor occupying the active site. In this study, structures ...

    Abstract | Full Text HTML | Hi-Res PDF | PDF w/ Links

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