The substrate binding site of human liver cytochrome P450 2C9: an NMR study.
Purified recombinant human liver cytochrome P450 2C9 was produced, from expression of the corresponding cDNA in yeast, in quantities large enough for UV-visible and 1H NMR experiments. Its interaction with several substrates (tienilic acid and two derivatives, lauric acid and diclofenac) and with a specific inhibitor, sulfaphenazole, was studied by UV-visible and 1H NMR spectroscopy. At 27 degrees C, all those substrates led to an almost complete conversion of CYP 2C9 to high-spin (S = 5/2) CYP 2C9-substrate complexes characterized by a Soret peak at 390 nm; their KD values varied between 1 and 42 microM. On the contrary, sulfaphenazole led to a low-spin (S = 1/2) CYP 2C9 complex upon binding of its NH2 group to CYP 2C9 iron. Interactions of the five substrates with the enzyme were studied by paramagnetic relaxation effects of CYP 2C9-iron(III) on the 1H NMR spectrum of each substrate. Distances between the heme iron atom and substrate protons were calculated from the NMR data, and the orientation of the substrate relative to iron was determined from those distances. Finally, a model for substrate positioning in the CYP 2C9 active site was constructed by molecular modeling studies under the constraint of the iron-proton distances. It points out two structural characteristics for a compound to be selectively recognized by CYP 2C9: (i) the presence of an anionic site able to establish an ionic bond with a putative cationic residue of the protein and (ii) the presence of an hydrophobic zone between the substrate hydroxylation site and the anionic site. Sulfaphenazole was easily included in that model; its very high affinity for CYP 2C9 is due to a third structural feature, the presence of its NH2 function which binds to CYP 2C9 iron.
Poli-Scaife
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Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, URA 400 CNRS, Université Paris V, 45 Rue des Saints-Pères, 75270 Paris Cedex 06, France.
Attias
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Dansette
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Mansuy
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004008:Diclofenac / Q000378:metabolism; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D051381:Rats; D013053:Spectrophotometry; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013426:Sulfaphenazole / Q000737:chemistry / Q000378:metabolism; D013989:Ticrynafen / Q000378:metabolism
D007850:Lauric Acids; D013426:Sulfaphenazole; C030358:lauric acid; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D013989:Ticrynafen