To probe whether the interaction of Cox11 and HKI with the LD of RanBP2 modulates the enzymatic activity of HKI, we first examined the effect of increasing concentrations of Cox11 on the initial rates of HKI enzymatic activity (Figure 2A). Cox11 strongly inhibits HKI activity in a concentration-dependent fashion, and at ~15 nM of Cox11, HKI activity could not be recorded (Figure 2A). Cox11 behaves as a partial noncompetitive inhibitor of HKI by affecting the V max of HKI for glucose (Figure 2B). Then, we evaluated the effect of the LD of RanBP2 on the HK activity in the presence of a fixed inhibitory concentration of Cox11, saturating concentration of glucose substrate, and increasing concentrations of LD. As shown in Figure 2C, the LD domain sharply reversed the inhibitory effect of Cox11 on HKI activity in a concentration-dependent manner, but under saturating (and stochiometric) amounts of LD, the velocity of the reaction did not reach that observed for HKI activity in the absence of Cox11 (Figure 2A), suggesting the LD by itself may also have an effect on HKI activity. Indeed, a saturating concentration of LD reduced the V max but not the K m of HKI (Figure 2D) by ~20% under similar conditions.