Inhibition of bovine hepatic fructose-1,6-diphosphatase by substrate analogs.

Purified bovine hepatic fructose-1,6-diphosphatase, which exhibits maximal activity at neutral pH, is competitively inhibited by several analogs of its substrate, fructose 1,6-diphosphate. These include glucose 1,6-diphosphate (Ki = 9.4 X 10(-5) M), hexitol 1,6-diphosphate (Ki = 2.3 X 10(-4) M), and 2,5-anhydro-D-mannitol 1,6-diphosphate (Ki = 3.3 X 10(-8) M), and 2,5-anhydro-D-glucitol 1,6-diphosphate (Ki = 5.5 X 10(-7) M). The Ki values for both 2,5-anhydro-D-mannitol 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate are lower than the Km of 1.4 X 10(-6) M for fructose 1,6-diphosphate. Since 2,5-anhydro-D-mannitol 1,6-diphosphate is an analog of the beta anomer of fructose 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate is an analog of the alpha anomer, the lower Ki for the mannitol analog may indicate that the beta anomer of fructose 1,6-diphosphate, which predominates in solution, is the true substrate. The substrate analog 1,5-pentanediol diphosphate inhibits slightly (K0.5 = 5 X 10(-3) M), but 1,4-cyclohexyldiol diphosphate does not. The Ki for product inhibition by sodium phosphate is 9.4 X 10(-3) M. 2,5-Anhydro-D-mannitol 1,6-diphosphate and alpha-D-glucose 1,6-diphosphate are substrates at pH 9.0, but not at pH 6.5.     

Hexose diphosphates and phosphofructokinase in rat brain during development

Fructose 2,6-diphosphate and glucose 1,6-diphosphate concentrations were determined during late gestation and over the course of suckling in rat brain cortex and cerebellum. Cortex fructose 2,6-diphosphate concentration was greatest in neonatal animals and gradually declined thereafter by 25% to reach the adult level at 15 days of age. In contrast, the glucose 1,6-diphosphate concentration increased 4-fold over the same period to reach its highest level by postnatal day 15. Neither cerebellar fructose 2,6-diphosphate nor glucose 1,6-diphosphate concentrations varied significantly. Six day cortex 6-phosphofructo-1-kinase was less sensitive to inhibition by citrate than the enzyme obtained from 15 day pups, and fructose 2,6-diphosphate was better than glucose 1,6-diphosphate at relieving the inhibition imposed by citrate at either age. It is suggested that the rise in cerebral glucose use which occurs during suckling cannot be attributed to either changes in the concentrations of fructose 2,6-diphosphate or glucose 1,6-diphosphate, or the age-related differential sensitivity of 6-phosphofructo-1-kinase toward these effectors.     

The role of fructose-1, 6-diphosphate in cell migration and proliferation in an in vitro xenograft blood vessel model of vascular wound healing

Summary Both smooth muscle cells and endothelial cells play an important role in vascular wound healing. To elucidate the role of fructose-1, 6-diphosphate, cell proliferation and cell migration studies were performed with human endothelial cells and rat smooth muscle cells. To mimic blood vessels, endothelial and smooth muscle cells were used in 1:10, 1:5, and 1:1 concentrations, respectively, mimicking large-, mid-, and capillary-sized blood vessels. Cell migration was studied with fetal bovine serum-starved cells. For cell proliferation assay, cells were plated at 30–50% confluency and then starved. The cells were incubated for 48 h with fructose-1, 6-diphosphate at (per ml) 10 mg, 1 mg, 500 μg, 250 μg, 100 μg, and 10 μg, pulsed with tritiated-thymidine and incubated with 1 N NaOH for 30 min at room temperature, harvested, and counted. For migration assay, confluent cells were starved, wounded, and incubated for 24 h with same concentrations of fructose-1, 6-diphosphate as in proliferation assay. The cells were fixed and counted. Smooth muscle cell proliferation was inhibited by fructose-1, 6-diphosphate at 10 mg/ml. In the xenograft models of 1:10, 1:5, and 1:1 fructose-1, 6-diphosphate inhibited proliferation at 10 mg/ml. In migration studies 10 mg fructose-1, 6-diphosphate per ml was inhibitory to both cell types. In large-, mid-, and capillary-sized blood vessels, fructose-1, 6-diphosphate inhibited proliferation of both cell types at 10 mg/ml. At the individual cell level, fructose-1, 6-diphosphate is nonstimulatory to proliferation of endothelial cells while inhibiting migration, and it acts on smooth muscle cells by inhibiting both proliferation and migration.     

The Formation of Glucose 1,6-Diphosphate from Fructose 1,6-Diphosphate by Yeast Phosphoglucomutase

It was found that fructose 1,6-diphosphate, the main intermediate of glycolysis, was able to act as a coenzyme of yeast phosphoglucomutase reaction. The mechanism of the coenzymatic activity of fructose 1,6-diphosphate was studied. It was indicated in the fructose 1,6-diphosphate dependent reaction that glucose 1,6-diphosphate was formed by the phosphate-transfer of fructose 1,6-diphosphate to glucose 1-phosphate in the first step, and in the second step the conversion of glucose 1-phosphate to glucose 6-phosphate, the original mutase reaction, occurred in the presence of glucose 1,6-diphosphate. The kinetic constants in the reaction of the first step were determined from the time courses of the fructose 1,6-diphosphate dependent reaction.     

Tagatose-1,6-diphosphate activation of lactate dehydrogenase from Streptococcus cremoris

Tagatose-1,6-diphosphate was an effective substitute for fructose-1,6-diphosphate in the activation of lactate dehydrogenase (EC 1.1.1.27) from $\text{Streptococcus cremoris}$ AM2. The K_m for pyruvate, V_max and 0.5 values (activator concentration at half-maximal velocity) were similar with each activator. Of the other sugar phosphates and glycolytic intermediates tested only glucose-1,6-diphosphate activated the enzyme although the 0.5 value was 200 times that for the ketohexose diphosphates. Lactate dehydrogenases from several other organisms belonging to the Lactobacillaceae were equally stimulated by fructose-1,6-diphosphate and tagatose-1,6-diphosphate.     

Anomeric specificity of fructosediphosphate aldolase E.C.4.1.2.13 from rabbit muscle

Fructosediphosphate aldolase from rabbit muscle is shown to accept β-D-fructofuranose-1,6-diphosphate as substrate, whereas α-D-fructofuranose-1,6-diphosphate can only be cleaved by the enzyme after a spontaneous change of configuration. The first order velocity constant of the spontaneous reaction was computed to be 0.55 sec^?1 (at 25° C, pH 7.6). The equilibrium mixture of D-fructose-1,6-diphosphate was computed to 26% α- and 74% β-D-fructofuranose-1,6-diphosphate.     

A comparison of the properties of the pyruvate kinases of the fat body and flight muscle of the adult male desert locust

1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1mum) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25 degrees for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1-2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7.2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7.2 as with the fat-body enzyme and the other at about pH8.5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8.5 and above.     

Modification of Human Erythrocyte Pyruvate Kinase by an Active Site-directed Reagent: Bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5′s-diphosphate, phospho-enolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, k_s, was 1.84 × 0.15 min^?1. K_d of the bromopyruvate-enzyme complex was 0.14 × 0.03 mM.

The presence of adenosine-5′-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5′-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.     

Modification of human erythrocyte pyruvate kinase by an active site-directed reagent: bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Nucleotide regulation of phosphoenolpyruvate carboxylase from Escherichia coli

Adenine, cytosine, guanine, and uracil nucleotides were surveyed as possible modulators of Escherichia coli phosphoenolpyruvate carboxylase. CMP, CDP, CTP, GDP, and GTP activate, ATP and GMP inhibit. The other nucleotides are without effect. Nucleotide activation is synergistic with acetyl-CoA or laurate. Cytosine nucleotide activation is also synergistic with fructose 1,6-diphosphate, whereas guanine nucleotide activation is not. The pH profiles for CMP and GDP activation, studied individually between pH 7.0 and 9.0, are similar to those for activation by fructose 1,6-diphosphate. ATP inhibits activation by acetyl-CoA, laurate, or fructose 1,6-diphosphate. Pairs of activators synergistically relieve the inhibition. Acetyl-CoA with laurate is most effective. Energy charge profiles suggest little sensitivity to charge fluctuation near 0.8. Ribose 5-phosphate also inhibits activation by acetyl-CoA, laurate, or fructose 1,6-diphosphate. GMP selectively inhibits fructose 1,6-diphosphate activation.     

Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-disphosphate and methods for characterizing hysteretic transitions

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, k(app.)=0.24s(-1) with 0.02mm-fructose 1,6-diphosphate (t(0.9) approximately 10s where t(0.9) is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20mum). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t(0.9)=0.5-1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.     

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers

Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.     

Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12

Summary We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.Abbreviations G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1-6DP fructose-1,6-diphosphate - PEP phosphoenol pyruvate     

Studies on the interaction between rabbit liver pyruvate kinase and its allosteric effector fructose 1,6-diphosphate

Preparation of the L form of rabbit liver pyruvate kinase (EC 2.7.1.40) in the presence of fructose 1,6-diphosphate yielded an enzyme which was kinetically identical with the M or muscle-type form of pyruvate kinase found in liver. Chromatographic and dialysis studies of this complex showed that most of the fructose 1,6-diphosphate molecules were loosely bound to the enzyme, but dilution-dissociation studies and binding experiments established that there was a high initial affinity between the enzyme and fructose 1,6-diphosphate (K(assoc.)=2.3x10(9)), and that binding of the loosely bound fructose 1,6-diphosphate was concentration-dependent and a necessary condition to overcome the co-operative interaction observed with the homotropic effector phosphoenolpyruvate. Preparation of the liver enzyme in the absence of EDTA did not yield a predominantly M form of the enzyme, and incubation of the M form in the presence of EDTA did not convert it into the L form, but resulted in inhibition of enzyme activity. Immunological studies confirmed that the L and M forms in liver were distinct, and that preparation of the L form in the presence of fructose 1,6-diphosphate did not produce an enzyme antigenically different from the L form prepared in the absence of this heterotropic effector.     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Inhibition of protein synthesis by glucose 6-phosphate and fructose 1,6-diphosphate in lysed rabbit reticulocytes and the reversal of inhibition by NAD+.

Glucose 6-phosphate and fructose 1,6-diphosphate inhibit protein synthesis when added to lysed rabbit reticulocytes. Protein synthesis is inhibited 47% with 6 mM fructose 1,6-diphosphate and 86% with 6 mM glucose 6-phosphate. With 0.125 mM NAD⁺, the inhibitory effect of glucose 6-phosphate and fructose 1,6-diphosphate becomes stimulatory. The stimulation of protein synthesis in those assays with NAD⁺ and the phosphorylated sugars is 50% above those assays that contain NAD⁺ alone. The inhibition of protein synthesis by glucose 6-phosphate and the reversal of this inhibition by NAD⁺ occurs at a step before the synthesis of the initial dipeptide, methionyl-valine. These data illustrate the importance of NAD⁺ and the activation of glycolysis in regulating protein synthesis in lysed rabbit reticulocytes.     

Purification and properties of dihydroxyacetone kinase from Klebsiella pneumoniae.

Dihydroxyacetone (DHA) kinase of Klebsiella pneumoniae, a gene product of the dha regulon responsible for fermentative dissimilation of glycerol and DHA, was purified 120-fold to a final specific activity of 10 mumol X min-1 X mg of protein-1 at 30 degrees C. The enzyme, a dimer of a 53,000 +/- 5,000-dalton polypeptide, is highly specific for DHA (Km, ca.4 microM). Glycerol is not a substrate at 1 mM and is not an inhibitor even at 100 mM. The enzyme is not inhibited by 5 mM fructose-1,6-diphosphate. Ca2+ gives a higher enzyme activity than Mg2+ as a cationic cofactor. Escherichia coli glycerol kinase acts on both glycerol and DHA and is allosterically inhibited by fructose-1,6-diphosphate. Antibodies raised against E. coli glycerol kinase cross-reacted with K. pneumoniae glycerol kinase but not with K. pneumoniae DHA kinase.     

Anaerobic expression of maize fructose-1,6-diphosphate aldolase

The anaerobic proteins of maize are a set of 10 major and 10 minor polypeptides selectively synthesized in anaerobic seedling roots. 1) Anaerobiosis resulted in the selected labeling of a protein which bound to Blue Sepharose and was eluted by fructose 1,6-diphosphate. 2) This protein elicited antiserum which recognized a single protein with molecular weight of approximately 40,000. 3) By Western blot analysis, this antiserum recognized a maize fructose-1,6-diphosphate aldolase purified to homogeneity. We show that two major anaerobic proteins of maize, ANP35.5 and ANP33A, correspond to a cytoplasmic fructose-1,6-diphosphate aldolase.