Mutational Analysis of VKOR.

More than 21 million prescriptions for warfarin are written yearly in the US for Vitamin K epoxide reductase (VKOR), the target of warfarin The vitamin K epoxide reductase, VKOR, apparently uses cysteines, 132 and 135 as active sites. In addition to cysteines 132 and 135, cysteines 43 and 51 are conserved throughout evolution. Rost et al. have mutated each of the cysteines in VKOR and found that, in whole cell lysates, mutations in of C43 result in less than 20% of wild-type activity while mutation of C53 eliminates activity. We have repeated these experiments and mutated all of the cysteine residues in VKOR. Our results in microsomes are similar to the results of Rost et. al. (2005) Thromb. Haemost. 94, 780–786. However, when the mutated enzymes are purified we find that the activity of C43 has 30% residual activity while C51 has 60% residual activity. Mutation of both residues in the same molecule results in an enzyme that is similar to the C51 mutation. In addition, we find that a portion of purified VKOR has a disulfide bond between residues 43 and 51. This suggested that we might be able to remove the loop between C43 and C51 and retain activity. Indeed, the mutated enzyme with this loop removed also has substantial activity. Warfarin inhibition studies suggest that these mutations do not materially affect warfarin sensitivity. Our results stress the importance of utilizing purified enzyme for interpreting the results of mutations in VKOR.