Enzyme Kinetics Of Cytochrome P450 In Catalytic Cycle

In the study of eADME in drug discovery, it was widely reported that the error of the kinetic parameters in the drug evaluation in vitro induced the blowing-up departure of the predictive values (Clint, intrinsic clearance ratio) from the real values of drug metabolism in vivo. Cytochrome P450 (CYP), the essential enzymes in drug metabolism, exhibited the atypical kinetics to confound our prediction of drug clearance. Increasing studies extrapolated that homotropic and heterotropic phenomena of CYP enzymes could be encountered not only in substrate activation but also in productive coupling of the electron donors like cytochrome P450 reductase (CPR) and cytochrome B5, and the lipid environment of the system also appeared to play a role as an effector. In this work, we focused on the investigation of the interaction of CYP, CPR, and lipid membrane in simple and complex systems, to extrapolate the influence of the components in catalytic cycle on the CYP kinetics, further to modify the previously supposed models on the atypical kinetic phenomena of CYP enzyems.First, ten CYP genes and CPR gene were obtained. Then a new Saccharomyces cerevisiae system coexpressed CPR and CYP3A4 was estabilished, which was named by BWG-CPR/3A4. Results showed that the system was stable and efficient to evaluate the drug metabolism in vitro. The metabolic activity of CYP3A4 in yeast was similar to the CYP3A4 activity in vivo.Second, BWG-CPR/3A4 was applied to study the function of two new mutagenesis of N-terminal fragment of CYP3A4. The activities of mutant enzymes and wild type enzymes were analyzed by the 6β-testosterone hydroxylation and nifedipine oxidation. Besides, ketoconazole and CPR antibodywere selected as the potential anchoring inhibitor to inhibit CYP3A4 activities. The results suggested that Arg 11 of CYP3A4 might be the potential site to bind with microsome membrane.Third, human CPR was expressed in E.coli. Recombinant protein was purified and anti-hCPR IgG was prepared. We compared the CPR kinetics in E.coli system, S.cerevisiae system and human liver microsome. The allosteric kinetics of CPR was discovered in yeast system. The possible mechanism of the kinetic phenomena was explained by the two-substrate kinetic model and the interaction with phospholipids membrane.Following the assumption of CPR influencing on CYP kinetics, we discovered the allosteric kinetics of CYP2A6 in recombinant system, which were potentially regulated by CPR. Moreover, ergosterol amount in yeast microsome was analyzed to be correlated with CPR kinetics.Finally, the allosteric kinetics of CPR was supposed to be correlated with the liver diseases, which was expected to be the biological marker to symbol the liver condition.