Effector-mediated alteration of substrate orientation in cytochrorne P4502C9

Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4'-hydroxylation is activated by the presence of dapsone resulting in reduction of the K-m, for flurbiprofen hydroxylation and an increase in V,m. Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in K-S) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T-1 relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 muM CYP2C9 and 145muM flurbiprofen in the presence and absence of 100muM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 Angstrom in the absence of dapsone and from 3.2 to 3.8 Angstrom in the presence of dapsone. The 4' proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 Angstrom, as compared to the absence of dapsone, 4.41 Angstrom. Dapsone protons were less affected, being 4.40 Angstrom from the heme in the absence of flurbiprofen and 4.00-4.01 Angstrom from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4' proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.