Abstract
Cytochrome P450 3A4, a major drug-metabolizing enzyme in man, is well known to show non-Michaelis-Menten steady-state kinetics for a number of substrates, indicating that more than one substrate can bind to the enzyme simultaneously, but it has proved difficult to obtain reliable estimates of exactly how many substrate molecules can bind. We have used a simple method involving studies of the effect of large inhibitors on the Hill coefficient to provide improved estimates of substrate stoichiometry from simple steady-state kinetics. Using a panel of eight inhibitors, we show that at least four molecules of the widely used CYP3A4 substrate 7-benzyloxyquinoline can bind simultaneously to the enzyme. Computational docking studies show that this is consistent with the recently reported crystal structures of the enzyme. In the case of midazolam, which shows simple Michaelis-Menten kinetics, the inhibitor effects demonstrate that two molecules must bind simultaneously, consistent with earlier evidence, whereas for diltiazem, the experiments provide no evidence for the binding of more than one molecule. The consequences of this “inhibitor-induced cooperativity” for the prediction of pharmacokinetics and drug-drug interactions are discussed.
Footnotes
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This study was supported by the Drug Metabolism Consortium (AstraZeneca, Aventis, Boehringer-Ingelheim, Celltech Chiroscience, GlaxoSmithKline, Hoffman-La Roche, Johnston and Johnston Pharmaceuticals, Merck Sharp and Dohme, Novartis, Novo Nordisk, Pfizer, Pharmacia, and Wyeth) and by the Higher Education Reach-Out to Business and the Community Fund.
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Y.K. and M.J.I.P. contributed equally to this work.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.021733.
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ABBREVIATIONS: P450, cytochrome P450; 7BQ, 7-benzyloxyquinoline; S0.5, concentration of substrate giving half maximal rate of catalysis; nH, Hill coefficient; HLM, human liver microsome; PDB, Protein Data Bank.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. -
↵1 Current affiliation: CXR Biosciences, James Lindsay Place, Dundee Technopole, Dundee, UK.
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↵2 Current affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK.
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↵3 Current affiliation: Chemistry Department, University Autonomous of Barcelona, Bellaterra, Catalonia, Spain.
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↵4 Current affiliation: Manchester Interdisciplinary Biocentre and School of Chemical Engineering and Analytical Science, University of Manchester, Manchester, UK.
- Received April 4, 2008.
- Accepted July 17, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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