Structural determinants of CYP2C9's genetic variability, substrate specificity and dioxygen cleavage.

摘要

Cytochrome P450 2C9 is a major form of human liver P450 that metabolizes approximately 15% of marketed drugs. Unexpected interruptions in CYP2C9 activity, due to drug-drug and drug-gene interactions, can cause adverse drug interactions which detailed knowledge of the structure-activity relationships for the enzyme might avoid. Therefore, the purpose of this dissertation is to advance our knowledge of structure-function relationships of CYP2C9 through studies with naturally occurring single nucleotide polymorphisms (SNPs) and rationally designed mutant forms of the enzyme. In term of genetic variation, examination of several recently described ethnic-specific polymorphisms (Chapter 3), highlighted the need for rigorous SNP validation prior to association studies on clinical or disease states, because the I181L, H184P, Q192P and L208V 'polymorphisms' were found to be spurious as a result of inadequate primer design in the original study. The objective of Chapter 2 was to investigate the mechanism underlying reduced enzyme activity of CYP2C9.11 which is encoded by the CYP2C9*11 (R335W) allele. It was shown that decreased stability and/or improper folding of the enzyme is responsible for reduced (S)-warfarin activity of the variant enzyme---the first example of this type for CYP2C9. Chapter 4 describes active site re-engineering studies for CYP2C9 involving the critical active site residue, R108, intended to rationally re-engineer CYP2C9 to accept basic ligands. As expected, the charge reversal mutant, R108E, was able to metabolize pyrene, an uncharged molecule, but lost catalytic activities towards the acidic substrate, (S)-warfarin and diclofenac. The R108E/D293N double mutant displayed a CYP2D6-like regioselectivity towards the basic substrate, propranolol, but with low overall catalytic efficiency. These studies demonstrated the critical role of these two amino acids for charge-pairing interactions in the CYP2C9 active site, but suggested the need for more extensive re-engineering to achieve substantial modifications to substrate specificity. In Chapter 5, replacement of up to seven active site amino acids of CYP2C9 with the corresponding residues from CYP2C19 provided enzymes with greatly altered ligand specificities. The mutants also exhibited a preference for heterolysis versus homolysis of BHTOOH, thereby mimicking the peroxyquinol ligand interaction with CYP2C19, possibly due likely to modified active site topology and hydration status.