Biphasic effect of fructose 2, 6-bisphosphate on the liver fructose-1, 6-bisphosphatase: mechanistic and physiological implications (original) (raw)
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Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-biphosphate
Proceedings of the National Academy of Sciences, 1981
Biochem. J. 192, 897-901] was found to inhibit, at micromolar concentrations, liver and muscle fructose-1,6bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11). The main characteristics of this inhibition are 'that (i) it is much stronger at low than at high substrate concentrations, (ii) it changes the substrate saturation curve from almost hyperbolic to sigmoidal, and (iii). it is synergistic with the inhibition by AMP. This inhibition may play an important role in the stimulation of gluconeogenesis by glucagon, because this hormone is known to decrease the concentration of fructose 2,6-bisphosphate in the liver [
Fructose-2,6-bisphosphatase from rat liver
European journal of biochemistry / FEBS, 1982
An enzyme that catalyzes the stoichiometric conversion of fructose 2,6-bisphosphate into fructose 6-phosphate and inorganic phosphate has been purified from rat liver. This fructose 2,6-bisphosphatase copurified with phosphofructokinase 2 (ATP: D-fructose 6-phosphate 2-phosphotransferase) in the several separation procedures used. The enzyme was active in the absence of Mg2+ and was stimulated by triphosphonucleotides in the presence of Mg2+ and also by glycerol 3-phosphate, glycerol 2-phosphate and dihydroxyacetone phosphate. It was strongly inhibited by fructose 6-phosphate at physiological concentrations and this inhibition was partially relieved by glycerol phosphate and dihydroxyacetone phosphate. The activity of fructose 2,6-bisphosphatase was increased severalfold upon incubation in the presence of cyclic-AMP-dependent protein kinase and cyclic AMP. The activation resulted from an increase in V (rate at infinite concentration of substrate) and from a greater sensitivity to th...
The interaction of fructose 2,6-bisphosphate and AMP with rat hepatic fructose 1,6-bisphosphatase
Journal of Biological Chemistry, 1983
The binding of the inhibitory ligands fructose 2,6bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-Pz and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperativity as indicated by K'1> K'z > K't 2 K ' 4 and a Hill plot, the curvature of which indicates K ' Z / K ' I c 1, K'3/K'z < 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K ' l c K f 2 < K'3 < K'4 and an nH
The Journal of biological chemistry, 1994
The effect of glucose on hepatic fructose (Fru) 2,6-P2 in starved rats was investigated. When livers were perfused with high glucose (40 mM), hexose-P in the liver increased immediately reaching the maximum within in 2 min, but Fru 2,6-P2 after a lag period of 4 min increased linearly. The activation of Fru 6-P,2-kinase and inactivation of Fru 2,6-Pase also showed a similar lag period. Determination of the phosphate contents of the bifunctional enzyme after 10 min of glucose perfusion revealed that 90% of the enzyme was in the dephospho form while only 10% of the control liver enzyme was dephosphorylated. Comparison of crude extracts of liver perfused with either high glucose or normal glucose (5.6 mM) showed that high glucose livers contained 50% higher protein phosphatase activity, which dephosphorylated the bifunctional enzyme. Subcellular fractionation of the extract showed that activation of the protein phosphatase occurred in the cytosol. Desalting of the cytosolic fraction re...
Purification of human liver fructose-1,6-bisphosphatase
Biochimica et biophysica acta, 1980
Human liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been purified 1200-fold using a heat treatment step followed by absorption on phosphocellulose at pH 8 and specific elution with buffer containing the substrate (fructose 1,6-bisphosphate) and allosteric effector (AMP). The enzyme is homogeneous in electrophoresis in polyacrylamide gel, in the presence and absence of denaturing agent. It has a molecular weight of 144 000 and is composed of four identical or nearly identical subunits. Fluorescence spectra indicate that the enzyme does not contain tryptophan residues. The pH optimum is 7.5 and the Km is determined as 0.8 microM. The enzyme is inhibited by AMP in cooperative manner with a K0 x 5 of 6 microM.
Kinetic studies of bovine liver fructose-1,6-bisphosphatase
The Journal of biological chemistry, 1979
Initial rate kinetic studies with bovine liver fructose-1,6-bisphosphatase were carried out in both directions of the reaction to determine the sequence of product release from the enzyme. Product inhibition by fructose-6-P was found to be S-linear, I-linear noncompetitive relative to fructose-1,6-bisphosphate, whereas inorganic orthophosphate was determined to be linear competitive with respect to the substrate. The kinetics of the reverse reaction were studied by coupling the phosphatase reaction to the aldolase, triosephosphate isomerase, and glycerolphosphate dehydrogenase reactions. The kinetic results were found to be in harmony with the Uni Bi ordered and random sequential mechanisms as well as a Uni Bi ping-pong mechanism. The nomenclature is that of Cleland (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137). However, nonkinetic considerations, when taken together with the kinetic results, suggest that the steady state ordered Uni Bi mechanism is the most likely possi...
Journal of Biological Chemistry
Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two min...
Molecular and Cellular Endocrinology, 1982
Fructose 2,6-bisphosphate was discovered in the course of studies on the regulation of hepatic 6-phosphofructo-1-kinase activity by glucagon. The compound is acid-labile but stable to heating in alkali. These properties are due to the presence of a phosphate on the hemiketalic hydroxyl group at C2. Fructose 2,6_bisphosphate can be synthesized chemically by reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide followed by base-catalyzed ring opening of the fructose 1,2-cyclic, 6-bisphosphate intermediate. Fructose 2,6_bisphosphate is a potent allosteric activator of 6-phosphofructo-1-kinase and an inhibitor of fructose 1,6bisphosphatase. It potentiates the effect of AMP on both enzymes. Since 6-phosphofructo-lkinase is one of the most important control elements in glycolysis, it is likely that fructose 2,6-bisphosphate is a significant physiological regulator of this process in liver. Likewise, fructose 2,6-bisphosphate is probably a significant physiological regulator of fructose 1,6-bisphosphatase, a key gluconeogenic enzyme. For example, the effect of glucagon to enhance carbon flux through fructose 1,6-bisphosphatase and to inhibit flux through 6-phosphofructo-1-kinase in intact hepatocytes is explained by the ability of the hormone to lower the level of fructose 2,6-bisphosphate. This decrease in fructose 2,6-bisphosphate is brought about, at least in part, by a CAMP-dependent phosphorylation and inactivation of the enzyme responsible for its synthesis, 6-phosphofructo-2-kinase. This novel enzyme catalyzes the transfer of the y phosphate of ATP to the C2 position of fructose 6-phosphate. Phosphorylation of this enzyme is catalyzed by the CAMP-dependent protein kinase in vitro with concomitant inhibition of enzyme activity. Fructose 1,6-bisphosphatase and 6-phosphofructo-1-kinase are also substrates for the CAMP-dependent protein kinase both in vitro and in vivo. However, a role for phosphorylation in regulating their activity remains uncertain. The discovery of the unique sugar phosphate, fructose 2,6_bisphosphate, has been an important advance in our understanding of the regulation of carbohydrate metabolism in liver.