The cytochrome P450s metabolize a wide variety of xenobiotic and endogenous compounds. Biochemical, biophysical, and computational approaches are applied to elucidate the structure-function relationships which govern the interactions of P450s with substrates, inhibitors, membrane lipids, and microsomal proteins. Since these interactions modulate P450 activity, elucidation of their molecular mechanism will aid in (1) clarifying the mechanism of P450-mediated drug and carcinogen metabolism; (2) defining the role of individual P450s in the metabolism of endogenous and environmental chemicals; and (3) development of specific P450 inhibitors. The flash photolysis technique was used to examine the kinetics of CO binding to P450. P450s 3A4 and 1A1 exhibited nonexponential kinetics. The Maximum Entropy Method was applied to obtain a kinetic distribution profile that corresponds to the conformational landscape. This statistical approach yielded P450-specific conformer distributions that were altered by substrate addition. These results suggest that P450 structure may be best viewed as a distribution of different conformers. In contrast to P450s 1A1 and 3A4, monoexponential kinetics were observed for P450 2E1, which indicates a more homogeneous conformation. Homology modeling techniques were used to generate a molecular model of human P450 1A2. On the basis of active groups in the predicted substrate binding site, we computationally searched a small molecule database for complementary molecules. Several P450 inhibitors were identified by this approach. In addition, mutation of residues in this site modified the catalytic activity. These results thus provide support for our P450 homology model.
