INTERACTIONS OF SUBSTRATE AND PRODUCT WITH CYTOCHROME-P450 - P450(2B4) VERSUS P450(CAM)

In the present study, two P450s (P450(2B4) and P450(eam)) have been ex amined with regard to their interactions with their substrates and pro ducts utilizing the characteristic spectral perturbations as criteria for their binding. The results indicate that although there are differ ences between the two P450s (E) in regard to their precise interaction s with their substrates (S) and products CP), the spectral titration d ata were consistent with the two-site model-E + S <----> ES (K-1), E P <----> EP (K-2); EP + S <----> ESP (K-3); ES + P <----> ESP(K-4) in which S and P bind to E forming ESP. The data were inconsistent with the two-site model in which S and P compete for the same site. As requ ired by the two-site model, the relationship K2K3 = K1K4 was maintaine d with both P450s at all product concentrations tested, although K-3 a nd K-4 decreased considerably when product concentration was increased . The relationship K-3 >> K-4 was also maintained, indicating that wit h both enzymes' ESP is formed predominantly by binding of S to EP rath er than binding of P to ES, and that ESP dissociates predominantly to ES and P rather than EP and S. In other words, binding of S to EP faci litates the dissociation of P. This indicates that the relative parame ter values are compatible for ESP to have functional significance. The possible role of ESP in controlling catalytic rate and catalytic effi ciency is discussed. (C) 1998 Academic Press.