Significant variations in the metabolism of various drugs and environmental chemicals which are metabolized via the cytochrome P450 (CYP) enzymes exist between humans on an individual and population scale. Many of these interindividual variations are attributed to polymorphisms in the 2C subfamily of enzymes. CYP2C subfamily of enzymes are responsible for the metabolism of a number of therapeutic agents such as S-mephenytoin, omeprazole, proguanil, certain barbiturates, diazepam, propranolol, diclofenac, tolbutamide, imipramine, and taxol. The overall objectives of this work is elucidate the molecular and metabolic basis of CYP2C related polymorphisms, characterization of the substrate-structure metabolism relationships, and the determination of critical protein structures of CYP2C enzymes that infer substrate specificity and metabolic activity. Methods to study the metabolism and kinetics of the anticancer drug taxol and the anticonvulsant drug mephenytoin were developed in order to identify the polymorphisms, their functional significance, and the substrate recognition site (SRS) in CYP2C8 and CYP2C19. Cytochrome P450 2C8 (CYP2C8) is the main enzyme responsible for the metabolism of the anticancer drug palcitaxol (taxol). In the current study, 2 new CYP2C8 alleles were identified and contain coding changes: CYP2C8*2 has an Ile269 Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8. CYP2C8*2 was found only in African Americans at a frequency of 0.18. CYP2C8*3 was found primarily in Caucasians (0.13 frequency). The CDNs of both alleles as well as of CYP2C8*1 (wild-type) were expressed in E Coli and the metabolic activity of these enzymes toward taxol was assessed using HPLC. CYP2C8*3 was defective in the metabolism of taxol (turnover number was 15% of CYP2C8*1). CYP28*2 had a two fold higher Km and two fold lower intrinsic clearance towards taxol than the wild-type enzyme. A second study was conducted to identify critical amino acids that determine the specificity of human CYP2C19 for S-mephenytoin-4'-hydroxylation. Chimeras were constructed by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1-4 was active (30% of WT CYP2C19) indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin-4 '-hydroxylase (6% of the activity of WT CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin-4'-hydroxylation activity with a KM similar to that of CYP2C19 but a 3-fold lower K-cat. Three residues in helix I, S286N, V292A, and F2951, were essential for S-mephenytoin-4'-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.
