Vitamin K competition releases tightly bound warfarin. (A) Warfarin inhibition curves with different concentrations of purified VKORC1-warfarin complex and with K as the substrate. (B) Relative VKORC1 activity at varying concentrations of VKORC1-warfarin and K. (C) Warfarin inhibition of cellular activity of transfected VKORC1 under different K concentrations. The dashed line illustrates that, under the same warfarin concentration, VKORC1 activity is inhibited at 5 nM K but retained at 4 μM K. The double arrow indicates relative activity of the unknown enzyme (not inhibited by warfarin) at high warfarin concentration with 4 μM K. (D) Correlation between cellular warfarin IC₅₀ and K concentration (from panel C). (E) Relative activities of VKORC1 and the unknown enzyme in absence of warfarin. The total carboxylation activity from VKORC1 and the unknown enzyme is measured with endogenous level of VKORC1 (30 ng; Figure 2A) transfected into DKO cells. Activity of the unknown enzyme is measured without the VKORC1 transfection, and subtraction of this activity from the total activity gives the VKORC1 activity. The curves show Michaelis-Menten fittings, and the dashed line illustrates that activity of the unknown enzyme is considerably lower than that of VKORC1 at a high K concentration (4 μM).