Control of liver 6-phosphofructokinase by fructose 2,6-bisphosphate and other effectors.

Abstract

Rat liver 6-phosphofructokinase (ATP-D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was partially purified free of interfering enzymes by a rapid procedure. Fructose 2,6-bisphosphate, at micromolar concentrations, greatly stimulated the enzyme by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Its action was synergistic with that of AMP. As a stimulator of liver phosphofructokinase, fructose 2,6-bisphosphate was approximately 1000-and 2500-fold more efficient than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate, respectively. The concentration at which a half-maximal effect was obtained with the hexose bisphosphates was dependent upon the experimental conditions. It was relatively high at physiological concentrations of substrates, AMP, and Pi, and under these conditions the positive effect of fructose 1,6-bisphosphate was no longer detectable. This was probably due to the negative effect of fructose 1,6-bisphosphate as a reaction product inhibitor. It is concluded that fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate controls, in association with other effectors, the activity of phosphofructoldnase in the liver.There is a general agreement in the literature that 6-phosphofructokinase (ATP:D-fructose-6-phosphate l-phosphotransferase, EC 2.7.1.11) plays a major role in the control ofglycolysis in nearly all types of cells (1-6). The activity of this enzyme is controlled by several metabolites, most notably its two substrates, fructose 6-phosphate and ATP. The rate of the reaction is a sigmoidal function of fructose 6-phosphate concentration, and the saturation curve is shifted to the left in the presence of positive effectors, among which fructose 1,6-bisphosphate (7,8), the product of the reaction, and AMP are currently believed to play a major role. Negative effectors such as ATP and citrate have the opposite effect. The rate ofthe reaction plotted as a function of ATP concentration exhibits a typical inhibition by excess substrate. This inhibition is relieved by the positive effectors and by large concentrations of fructose 6-phosphate, whereas it is intensified by the negative effectors.Fructose 2,6-bisphosphate is a newly recognized, extremely effective, positive effector of liver and muscle phosphofructokinase (9). Its concentration in the liver is greatly increased under conditions in which glycolysis is active and is decreased by glucagon (10). It has also been found to inhibit, at micromolar concentrations, liver and muscle fructose-1,6-bisphosphatase (11). It therefore appears to be a major regulator of both glycolysis and gluconeogenesis in the liver. The purpose of the present work was to investigate the action of fructose 2,6-bisphosphate on the kinetics of liver phosphofructokinase and to compare it to that of its isomers fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and that of AMP. When the effect of fructose 1,6-bisphosphate was examined, pyruvate kinase (20 ,g/ml) and lactate dehydrogenase (25 Mg/ ml) were u...

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase

“…However, this effect was minuscule relative to the large decrease in the F2,6BP Ras and 6-phosphofructo-2-kinase S Telang et al of F2,6BP during immortalization may reflect a negative feedback compensation in response to increased flux at PFK-1, changes in F2,6BP stability in situ (e.g., low intracellular pH) or increased conversion of F2,6BP into F6P for glycolytic utilization. Nevertheless, these data run counter to the widely held assumption that the intracellular F2,6BP concentration directly corresponds to and possibly sets the proliferative rate (Van Schaftingen et al, 1981;Hue and Rousseau, 1993;Chesney et al, 1999). We observed marked differences in glucose metabolism between primary mouse fibroblasts and human bronchial epithelial cells during ras-transformation.…”

“…Flux at PFK-1 is suppressed by several allosteric effectors, including ATP (i.e., the Pasteur effect), H þ ions and citrate, which exert negative feedback when energy is abundant (Van Schaftingen et al, 1981). Oncogenic ras has not been found to decrease these intrinsic inhibitors, but rather to increase the steadystate concentration of an allosteric activator of PFK-1, fructose-2,6-bisphosphate (F2,6BP) (Kole et al, 1991).…”

“…F2,6BP activates PFK-1 by shifting the conformational equilibrium of PFK-1 from a low to a high-affinity state for its substrate, F6P. This increase in intracellular F2,6BP relieves the tonic inhibition of PFK-1 by ATP, allowing untethered glycolytic flux at the PFK-1 checkpoint to proceed (Van Schaftingen et al, 1981).…”

Regulation of hepatic altro heptulose 1,7‐bisphosphate levels and control of flux through the pentose pathway by fructose 2,6‐bisphosphate

“…The regulation of carbon flux through the hepatic fructose bisphosphatase-phosphofructokinase substrate cycle is regulated, in part, by the level of a novel sugar phosphate fructose 2,6-P2 [1][2][3][4][5][6][7]. Methods for its synthesis and its anomeric specificity have been reported [8,9].…”

Interactions of antibodies against soluble phosphofructokinase with the soluble and particulate enzymes from yeast

“…(EC 2.7.1.11) from yeast, a key enzyme of glucose catabolism [ 1,2], is composed of two kinds of subunits, LY andp, being present in a 1: 1 ratio and forming an octameric assembly [3,4]. It was deduced that the larger subunit LY does not participate in the catalytic process and the substrate binding sites being localized exclusively on the &subunits [5].…”