Biochemical and Mechanistic Studies of the Interactions Between Vitamin K Antagonists and Vitamin K Epoxide Reductase

Abstract

The studies that are presented in this dissertation have; (i) established a mechanism by which warfarin dose response is modulated by VKORC1 genotype under optimized conditions for kinetic analysis of human vitamin K epoxide reductase (VKOR) catalytic activity, (ii) evaluated previously suggested irreversible and reversible mechanisms of VKOR inhibition by vitamin K antagonists (VKAs), and (iii) elucidated the role of tyrosine 139 in the catalytic center and inhibitor binding site of VKOR. Firstly, observation of VKOR activity in human liver microsomes (HLMs) under varied experimental conditions demonstrated that human VKOR is a highly labile enzyme. Under optimal conditions, evaluation of genotyped HLMs for downstream effects of altered VKORC1 mRNA expression revealed a relationship between the VKORC1 B haplotype and increased the catalytic activity and protein expression of VKOR. This demonstrates that warfarin dose is regulated through a transcriptional mechanism wherein higher levels of VKORC1 mRNA translate to increased protein expression and associated enzymatic activity of VKOR, thus requiring a higher dose of warfarin to maintain the same target level of anticoagulation. Secondly, the co-existence of irreversible and reversible modes of VKOR inhibition was re-investigated by the dilution method with the addition of two treatments, 20 mM DTT and 4% BSA, aimed specifically at stringent removal of inhibitor from microsomes. A post-dilution evaluation of VKOR activity in bovine liver microsomes that had been treated with 4-hydroxycoumarin and 1,3-indanedione VKAs demonstrated that they are not irreversible inhibitors, but appear to be tight-binding compounds that exhibit non-competitive type reversible kinetics with respect to vitamin K epoxide (KO). Finally, a recombinantly expressed VKOR carrying a tyrosine→phenylalanine mutation, which was made to test the importance of Y139 as a base or proton donor in the mechanism of KO reduction, had no effect on steady-state kinetic parameters for the formation of vitamin K1 or 3-hydroxyvitamin K (a putative side-product). Any mutation of Y139, however, generated a warfarin-resistant enzyme. Therefore, although this residue is located proximally to the catalytic site of VKOR, we conclude that Y139 is not directly involved in the mechanism of substrate turnover, but has a pivotal role in warfarin binding and inhibition.