Warfarin inhibits full VKORC1 reduction and consequent carboxylation much more than expected from the inhibition of the 2 individual reactions. (A) Y139F was assayed under nitrogen for KO to KH₂ reduction in the absence (−) or presence (+) of warfarin. (B-D) Microsomes containing the carboxylase and Y139F or Y139H or wt VKORC1 were assayed for KO reduction to K, using high-performance liquid chromatography to monitor vitamin K forms, and for carboxylation by measuring [¹⁴C]-CO₂ incorporation into FLEEL. (E-F) The K to KH₂ reaction was analyzed by first assaying microsomes containing the carboxylase and wt VKORC1 (E) or Y139F (F) for KO to K inhibition, which gave the indicated concentrations of K. Carboxylation that depends on K to KH₂ reduction was then monitored for warfarin inhibition with these K concentrations, or with KO (65 µM).