Figure 3. α-ketoglutarate inhibits Enzyme I in vitro.

(a) Phosphoenolpyruvate (PEP) donates phosphate, which is transferred in turn to the cytosolic proteins EI, HPr, and EIIA^Glc, the membrane transporter EIIBC^Glc, and finally glucose, which is simultaneously transported and phosphorylated. All reactions except the final phosphorylation of glucose are reversible. (b) The activity of purified Enzyme I (EI) was determined in the presence (white squares) or absence (black squares) of 2 mM α-ketoglutarate. Plotted values are the estimated enzyme velocities ± standard error, and solid lines are the best fit Michaelis-Menten equation. Values are normalized to the estimated V_max in the absence of α-ketoglutarate. (c) EI activity was determined in the presence of 1 mM PEP and varying concentrations of α-ketoglutarate. The solid line is the best fit Hill equation for non-competitive inhibition. Values are normalized to the activity with no α-ketoglutarate added. (d) Cyclic-AMP (cAMP) concentration was monitored following a carbon downshift from glucose to glycerol at t = 0. Cells were grown on either 10 mM ammonia or 2.5 mM arginine; where indicated, 20 mM dimethyl-ketoglutarate was present in the glycerol media following downshift. Plotted values are the mean and standard error of three biological replicates.