Significant variations in the metabolism of 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 to elucidate the molecular and metabolic basis of CYP related polymorphisms, characterization of the substrate-structure metabolism relationships, and the determination of critical protein structures of CYP 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 CYP enzymes. CYP2C9 is a clinically important enzyme, responsible for the metabolism of numerous clinically important therapeutic drugs. Thirty eight single nucleotide polymorphisms in CYP2C9 were characterized by resequencing of genomic DNA from 92 individuals from three different racial groups. Haplotype analysis predicted that there are at least 21 alleles of CYP2C9 in this group of individuals. Six new alleles were identified that contained coding changes: L19I (CYP2C9*7), R150H (CYP2C9*8), H251R (CYP2C9*9), E272G (CYP2C9*10), R335W(CYP2C9*11) and P489S (CYP2C9*12). When expressed in a bacterial cDNA expression system, several alleles exhibited altered catalytic activity. CYP2C9*11 appeared to be a putative poor metabolizer allele, exhibiting a three-fold increase in the Km and more than a two-fold decrease in the intrinsic clearance for tolbutamide. Examination of the crystal structure of human CYP2C9 reveals that R335 is located in the turn between the J and J' helices and forms a hydrogen-bonding ion pair with D341 from the J' helix. Abolishing this interaction in CYP2C9*11 individuals could destabilize the secondary structure and alter the substrate affinity. This new putative poor metabolizer (PM) allele was found in Africans. A second potentially PM allele CYP2C9*12 found in a racially unidentified sample also exhibited a modest decrease in the Vmax and the intrinsic clearance for tolbutamide in a recombinant system. Further clinical studies are needed to determine the effect of these new polymorphisms on the metabolism of CYP2C9 substrates.
