CO binding kinetics of human cytochrome P450 3A4. Specific interaction of substrates with kinetically distinguishable conformers.

The kinetics of CO binding to human cytochrome P450 3A4 was examined by the flash photolysis technique, employing the membrane-bound P450 expressed in baculovirus-infected SF9 insect cells. Triexponential kinetics was observed, indicating that P450 3A4 is composed of multiple, kinetically distinguishable conformers. To define the substrate specificity of individual P450 3A4 conformers we evaluated the effect of a series of substrates of varying sizes and structures on the CO binding kinetics. The rate of CO binding to the total mixture of P450 3A4 conformers was increased in the presence of nifedipine and erythromycin, decreased by quinidine, testosterone, and warfarin, and unaffected by cimetidine and 17 alpha-ethynylestradiol. A recently developed kinetic difference method (Koley, A. P., Robinson, R. C., Markowitz, A., and Friedman, F. K. (1994) Biochemistry 33, 2484-2489) was used to define the kinetic parameters of individual P450 3A4 conformers. The results showed that different conformers have distinct substrate specificities. The substrates had markedly variable effects on the CO binding kinetics of their target P450 3A4 conformers and thus differentially modulate their conformations. These results demonstrate that the interaction of a particular substrate with a specific P450 3A4 conformer can be assessed in the presence of multiple conformers.