THE SUBSTRATE-BINDING SITE OF HUMAN LIVER CYTOCHROME-P450 2C9 - AN NMR-STUDY

Purified recombinant human liver cytochrome P450 2C9 was produced, fro m expression of the corresponding cDNA in yeast, in quantities large e nough for UV-visible and H-1 NMR experiments. Its interaction with sev eral substrates (tienilic acid and two derivatives, lauric acid and di clofenac) and with a specific inhibitor, sulfaphenazole, was studied b y UV-visible and H-1 NMR spectroscopy. At 27 degrees C, all those subs trates 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 3 90 nm; their K-D values varied between 1 and 42 mu M. On the contrary, sulfaphenazole led to a low-spin (S = 1/2) CYP 2C9 complex upon bindi ng of its NH2 group to CYP 2C9 iron. Interactions of the five substrat es with the enzyme were studied by paramagnetic relaxation effects of CYP 2C9-iron(III) on the H-1 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 w as determined from those distances. Finally, a model for substrate pos itioning in the CYP 2C9 active site was constructed by molecular model ing studies under the constraint of the iron-proton distances. It poin ts out two structural characteristics for a compound to be selectively recognized by CYP 2C9: (i) the presence of an anionic site able to es tablish an ionic bond with a putative cationic residue of the protein and (ii) the presence of an hydrophobic zone between the substrate hyd roxylation site and the anionic site. Sulfaphenazole was easily includ ed 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 C YP 2C9 iron.