ACTIVATION OF CYP3A4 - EVIDENCE FOR THE SIMULTANEOUS BINDING OF 2 SUBSTRATES IN A CYTOCHROME-P450 ACTIVE-SITE

A unique characteristic of the CYP3A subfamily of cytochrome P450 enzy mes is their ability to be activated by certain compounds. It is repor ted that CYP3A4-catalyzed phenanthrene metabolism is activated by 7,8- benzoflavone and that 7,8-benzoflavone serves as a substrate for CYP3A 4. Kinetic analyses of these two substrates show that 7,8-benzoflavone increases the V-max of phenanthrene metabolism without changing the K -m and that phenanthrene decreases the V-max of 7,8-benzoflavone metab olism without increasing the K-m. These results suggest that both subs trates (or substrate and activator) are simultaneously present in the active site. Both compounds must have access to the active oxygen, sin ce neither phenanthrene nor 7,8-benzoflavone can competitively inhibit the other substrate. These data provide the first evidence that two d ifferent molecules can be simultaneously bound to the same P450 active site. Additionally, structure-activity relationship studies were perf ormed with derivatives of 7,8-benzoflavone structure: The effects of 1 3 different compounds on the regioselectivity of phenanthrene, chrysen e, and benzo[a]pyrene metabolism were determined. Of the 13 compounds studied, 6 were activators, 2 Were partial activators, and 5 were inhi bitors. Analyses of the data suggest that (1) naphthalene substituted with a ketone in the 2-position can activate 3A4 and (2) the presence of an activator results in a narrower effective substrate binding site . Since the CYP3A enzymes are very important in drug metabolism, the p ossibility of activation, and autoactivation, must be considered when in vitro-in vivo correlations are made and when possible drug interact ions ace considered.