Effect of phosphoenolpyruvate on the activity of E. coli NADP-isocitrate dehydrogenase with respect to the threo-dS-isocitrate concentration. (A) threo-dS-Isocitrate saturation curves in the absence and presence of different concentrations of phosphoenolpyruvate. (B) Hill's plot of the data shown in panel A. Points represent experimental data, and lines are theoretically drawn by using the following equation: v = V · [S]ⁿ/([S]ⁿ + K_mⁿ) where [S] is the concentration of isocitrate, K_m is the concentration required for half-maximal velocity, and n is the Hill's interaction coefficient. Symbols: closed circles, no addition (K_m = 0.029 mM and n_H = 1.0); triangles, 0.2 mM PEP added (K_m = 0.046 mM and n_H = 1.1); squares, 1 mM PEP added (K_m = 0.08 mM and n_H = 1.1); open circles, 5 mM PEP added (K_m = 0.16 mM and n_H = 1.2).

Effect of phosphoenolpyruvate on the activity of E. coli isocitrate lyase with respect to the isocitrate concentration. (A) threo-dS-Isocitrate saturation curves in the presence of different concentrations of phosphoenolpyruvate. (B) Hofstee plot of the data in panel A. The Hofstee equation for uncompetitive inhibition is as follows: [S]/v = (1/V_max) · (1 + [I]/K_i′) · [S] + K_m/V_max. The relationship between [S]/v and [S], the substrate concentrations in the presence of various concentrations of PEP, the inhibitor, [I] gave straight lines converging on the ordinate at K_m/V_max. Points represent experimental data, and lines are theoretically drawn as demonstrated in Fig. 1 except that the n_H value was kept at 1. Symbols: open circles, no PEP (K_m = 0.89 mM and V_max = 6.72 μmol/min per mg protein); closed circles, 0.2 mM PEP added (K_m = 0.72 mM and V_max = 4.92 μmol/min per mg); triangles, 1 mM PEP added (K_m = 0.47 mM and V_max = 3.29 μmol/min per mg); squares, 5 mM PEP added (K_m = 0.10 mM and V_max = 0.894 μmol/min per mg).

Inhibition of E. coli NADP-isocitrate dehydrogenase by phosphoenolpyruvate. Reaction conditions were similar to those in the legend to Fig. 1, except the following modifications. The PEP concentrations were varied, and the threo-dS-isocitrate concentrations were fixed at 0.1 mM (circles), 0.02 mM (squares), and 0.004 mM (triangles). (A) Inhibition by phosphoenolpyruvate. (B) Variation of the quotient function Q with respect to the inhibition by PEP, assuming two binding sites for this inhibitor. The values of V′ taken in constructing this plot were determined from the velocity without PEP.