Research On The Relationship Of The Structure And The Catalysis Of Cytochrome P450 2C9

Cytochrome P450 2C9 (CYP2C9), a member of the CYP2C enzyme subfamily, ranks amongst the most important drug metabolizing enzymes in human. It makes up about 20% of the total Cytochrome P-450 protein in liver microsomes, and hydroxylates about 16% of drugs in current clinical use.Variation in drug toxicity and efficacy is consistently observed across patient populations. It contributes to environmental and genetic factors. Environmental factors contain irrational administrating, nutritional intake, smoking and drinking, etc. Genetic factors refer to a number of inherited sequence variants of genes encoding drug-metabolizing enzymes and drug receptors, which are the major cause of interindividual and ethnic variability.CYP2C9 allele, CYP2C9*13, caused significantly lower intrinsic clearance than wild-type. The reported X-ray crystal structure of CYP2C9 shows that leu90 almost exposed on the surface of CYP2C9, which is not the heme-binding region and is far from the substrate binding pocket.To study the role of different kinds of residues at position 90 in CYP2C9 catalytic activity, the Leu residue at position 90 was replaced by six amino acids, and their kinetic parameters were determined for catalyzing the following reactions of diclofenac 4'-hydroxylation, lornoxicam 5'-hydroxylation and P450-GloTM assays.We constructed three-dimensional models of the substrate-free CYP2C9 mutation proteins via molecular dynamic (MD) simulations, on the basis of the X-ray crystal structure of human CYP2C9.1, and did flexible docking of the mutants.The Vmax values for three substrates'hydroxylation of mutants L90P and L90E are lower than that of the wild-type CYP2C9 on contrast of the other five mutants. The energy minimization and molecular dynamics (MD) simulations showed that the entrance for substrates accessing shrank visibly after replacement of Leu by Glu and Pro, while the other mutants caused no significant changes compared to the wild-type. These constricted entrances blocked the access of substrates into the SRS, which is believed to be mainly mechanism of reduction in the catalytic activity in CYP2C9.13 and CYP2C9 L90E. The cavity size of all mutants decreased in MD, while the kinetic parameters were different among mutants. Another factor for enzymatic activity is the electrostatic potential distribution in the active site. The reduced interaction energies values reflect increased affinity, which, together with the changes in catalytic activity, suggests that residue90 has an important role on the interaction of enzyme and substrate. The thermal stability assay showed a similar tendency to the assays of the three different substrates.Our in-vitro results showed that the magnitude of CYP2C9*13-reduced drug metabolism is larger than that of CYP2C9*3. CYP2C9*13 homozygote individuals may face more risk of drug toxicity. The relevance of the various polymorphisms to drug response and toxicity is considered as well as the possibility that genotype for these polymorphisms may be a determinant for personalized prescribing in the future. In conclusion, the residue 90 has significantly effect on the catalytic activity of CYP2C9. The substitution at residue 90 affected the catalysis activity by the change in the size of the entrance, the shape of the pocket, the electrostatic potential distribution, and the thermal stability. And the mutants showed the significant alterations in both substrate binding capacity (Km) and enzymatic efficiency (Vmax), which suggests non-SRS amino acid affects significantly on the structures and activity, and suggests that changes of the whole enzyme structure have a more important effect on the activity. These new data on the relationship between structure and function of selected CYP2C9 mutants, and the result of our modeling experiments, should prove useful in future studies of analogue metabolism and drug design.In this work, we used PEI as the transfection reagent, and demonstrated PEI can transfect Sf9 insect cells. The cost of PEI reagent of transfected cells is very close to zero. The CPR and P450s were treated with CHAPS for reconstitution and optimization of catalytic activity. The result of diclofenac catalytic activity showed CYP2C9*13 had lower intrinsic clearance than CYP2C*3 due to more increase in Km and more decrease in Vmax. We established the baculovirus-insect cell system for the high-level expression of CYP2C9 and CPR successfully, which can conduct kinetic studies, to further characterize the catalytic activity of CYP2C9*13, explain the mechanism CYP2C9*13-mediated reduction of drug metabolism and the relationship between structure and function...