PubMed1981
In an attempt to clarify the kinetic properties of Escherichia coli phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] in vivo and to evaluate the physiological significance of the individual effectors, saturation curves were obtained for each ligand with reaction mixtures (pH 7.3) containing "physiological concentrations" of the other ligands in various combinations. As the "physiological concentrations" of ligands, which are defined as the concentrations of ligands found in the glucose-grown cells, the following values were employed: PEP, 0.2 mM; acetyl-CoA(CoA-SAc), 0.4 mM; fructose 1,6-bisphosphate(Fru-1,6-P2), 2.0 mM; GTP, 1.0 mM; L-aspartate, 1.0 mM; L-malate, 1.0 mM (Morikawa, M., Izui, K., Taguchi, M., & Katsuki, H. (1980) J. Biochem. 87, 441--449). In the absence of any activator the enzyme activity was very low. CoASAc was the most powerful activator. The other two activators (Fru-1,6-P2 and GTP) exhibited essentially no activation alone, but produced a strong synergistic activation with CoASAc. The severe inhibition by L-aspartate or L-malate was effectively alleviated only through this synergistic action of the activators. The presence of all three activators decreased the half-saturation concentration (S0.5) of PEP from 15 mM to 0.35 mM and increased the maximal velocity attainable at infinite concentration of PEP about 15-fold. In the system containing all five effectors, which is close to the in vivo condition, the saturation curve of PEP was sigmoidal with a Hill coefficient of 1.6 and with an S0.5 value of 3.0 mM, which is about 15-fold larger than its "physiological concentration." On the basis of the rate-concentration curve for each effector obtained with the reaction mixture containing PEP and the other effectors at "physiological concentrations," it was suggested that all five effectors significantly contribute to the enzyme activity in vivo. Palmitoleate, another activator of the enzyme, showed no activation in such a reaction mixture. The sensitivity of the enzyme to the "physiological concentration" of each effector was also observed in an in situ system using permeabilized E. coli cells, where the enzyme concentration was as high as in vivo.