EVALUATION OF ATYPICAL CYTOCHROME-P450 KINETICS WITH 2-SUBSTRATE MODELS - EVIDENCE THAT MULTIPLE SUBSTRATES CAN SIMULTANEOUSLY BIND TO CYTOCHROME-P450 ACTIVE-SITES

Some cytochrome P450 catalyzed reactions show atypical kinetics, and t hese kinetic processes can be grouped into five categories: activation , autoactivation, partial inhibition, substrate inhibition, and biphas ic saturation curves. A two-site model in which the enzyme can bind tw o substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 a nd naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as da psone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and napro xen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics su ggestive of a low K-m, low V-max component for the first substrate mol ecule and a high K-m, high V-max component for the second substrate mo lecule. 7,8-Benzoflavone activation of phenanthrene metabolism by CYP3 A4 and dapsone activation of flurbiprofen and naproxen metabolism by C YP2C9 were also observed. Furthermore, partial inhibition of 7,8-benzo flavone metabolism by phenanthrene was observed, These results demonst rate that various P450 isoforms may exhibit atypical enzyme kinetics d epending on the substrate(s) employed and that these results may be ex plained by a model which includes simultaneous binding of two substrat e molecules in the active site.