title: Multiple substrate binding by cytochrome P450 3A4: estimation of the number of bound substrate molecules.
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.
authors.affiliation: Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical, School, Dundee, United Kingdom.
authors.forename: Yury
authors.initials: Y
authors.lastname: Kapelyukh
authors.forename: Mark J I
authors.initials: MJ
authors.lastname: Paine
authors.forename: Jean-Didier
authors.initials: JD
authors.lastname: Maréchal
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sutcliffe
authors.forename: C Roland
authors.initials: CR
authors.lastname: Wolf
authors.forename: Gordon C K
authors.initials: GC
authors.lastname: Roberts
mesh_terms: D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D004110:Diltiazem / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D008874:Midazolam / Q000493:pharmacokinetics; D008958:Models, Molecular; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D004110:Diltiazem; D008874:Midazolam