Biosynthesis of Bacterial Glycogen XI. KINETIC CHARACTERIZATION OF AN ALTERED ADENOSINE DIPHOSPHATE-GLUCOSE SYNTHASE FROM A "GLYCOGEN-EXCESS" MUTANT OF ESCHERICHIA COLI B

Abstract Previous studies indicated that allosteric regulation of bacterial glycogen synthesis occurs at the level of ADP-glucose synthase (reviewed by Preiss, J. (1969) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds) Vol. 1, pp. 125–160, Academic Press, New York). The physiological significance of the allosteric effects observed in vitro is demonstrated by kinetic characterization of an altered ADP-glucose synthase from a glycogen-excess mutant of Escherichia coli B. The mutant enzyme is specifically altered in its responses to allosteric effectors, having a higher apparent affinity for activators (fructose-P2, TPNH, and pyridoxal-P) and a lower apparent affinity for inhibitor (AMP) than the wild type enzyme. These changes make the mutant enzyme more active at physiological energy charge values and are sufficient to account for the increased rate of glycogen accumulation in vivo. Previous studies demonstrated that allosteric activators increase the activity of the E. coli B ADP-glucose synthase by increasing its maximum velocity (4- to 8-fold) and increasing its affinity for substrates and metal ion (2- to 15-fold) (Priess, J., ibid). Activators also change the shape of the saturation curve for the substrate glucose 1-phosphate. The curves suggest that glucose-1-P binding sites are negatively cooperative in the absence of activators but noninteracting in their presence. A comparison of the concentrations of activators required to stimulate ADP-glucose synthase with their intracellular concentrations suggests that fructose-P2 is the physiological activator of the enzyme in E. coli.