Figure 5. Effect of mutations at position 189 on the affinity of EI for PEP and αKG.

(a) Michaelis-Menten kinetics for EI^WT (black), EI^Q (blue) and EI^A (red) with the substrate PEP. Experimental velocities were normalized to the maximum velocity obtained by the individual fits (solid lines). (b) Δ_H/N values for EI^WT (black), EI^Q (blue) and EI^A (red) as a function of αKG concentration. Data for all residues showing Δ_H/N > 0.1 ppm at 10 mM αKG were fit simultaneously using a one-site binding model per subunit (see Methods). The 3 datasets can be fit with the same K_D value (dashed line). In the figure, the Δ_H/N values were normalized respect to the fitted Δ_H/N at saturation and averaged out over all the residues used in the fitting procedure. The error bars are set to one standard deviation. (c,d) Structural models of PEP¹⁷ and αKG⁸, respectively, bound to the EIC domain. Ligands and side chains involved in the interactions are shown as sticks (carbon, grey; nitrogen, blue; oxygen, red; phosphorus, orange; sulfur, yellow). Backbone amides of N454 and D455 are shown as sphere (nitrogen, blue; hydrogen, white). The magnesium ion is displayed as a yellow sphere. β3α3 and β6α6 loops are colored cyan and pink, respectively (note that both β3α3 and β6α6 loops contain a short α-helical region). The molecular structures of PEP and αKG are shown on the left. (error bars: 1 s.d.)