Neurospora fructose-1,6-diphosphate aldolase: inhibition by sodium pyruvate

$\text{Neurospora}$ fructose-1,6-diphosphate aldolase exhibited a hyperbolic substrate saturation curve which changed to sigmoidal in the presence of 0.5 mM sodium pyruvate. The S_0.5 value for fructose-1,6-diphosphate increased from 1.4 mM to 6.6 and 20 mM in the presence of 0.5 and 1.0 mM sodium pyruvate, respectively. The inhibition seems to be cooperative in nature and involves conformational changes. Potassium ions completely blocked the inhibition by sodium pyruvate.     

Regulation of activation of ribulose bisphosphate carboxylase from Pseudomonasoxalaticus

6-phosphogluconate, potentiated activation of ribulose bisphosphate carboxylase from $\text{Pseudomonas}\text{oxalaticus}$ whereas fructose-1,6-bisphosphate inhibited activation and fructose-6-phosphate had no effect. The presence of 1 mM 6-phosphogluconate during activation reduced the K_act for Mg²⁺ from 1.4 mM to approximately 0.2 mM. In the absence of 6-phosphogluconate, the enzyme responded sigmoidally to increasing CO₂ (Hill coefficient, h, of 1.8), with a concentration causing half maximal activation, Act_0.5, of 15 mM NaHCO₃. In the presence of 1 mM 6-phosphogluconate h was reduced to 1.1 and an Act_0.5 value of 5 mM NaHCO₃ was obtained. 6-phosphogluconate appeared to saturate at or below 20 μmM.     

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.     

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.     

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     

Glucose- and mannose-1,6-P2 as activators of phosphofructokinase in red blood cells

Glucose-1,6-P₂ and mannose-1,6-P₂ are concluded to be important activators from “reconstruction” experiments showing that the other known effectors of phosphofructokinase poise it at ～ 0.1% of its V_max, compared with the $\text{in vivo}$ rate of ～ 1%. These activators may explain the relative insensitivity of red cell glycolysis to fructose-1,6-P₂. Glucose-1,6-P₂ is elevated more than two-fold in pyruvate kinase deficient cells but not in cells from patients with alkalosis although both have increased levels of the inhibitor, glycerate-2,3-P₂.     

Allosteric and isosteric modifiers of NADH binding to cytoplasmic malic dehydrogenase

The binding of NADH to cytoplasmic malic dehydrogenase is shown to be affected by a number of added ligands. One class of ligands appear to be analogs of a substrate for the enzyme, $\text{L}$-malate. These alter the binding constant for NADH without affecting the cooperativity of binding. In contrast, fructose-1,6-diphosphate behaves as an allosteric inhibitor at low enzyme concentrations, apparently by shifting the monomer-dimer equilibrium of the protein to the cooperatively binding dimer. The significance of these results are discussed in terms of a proposed regulatory function for the enzyme.     

Effect of vanadate on phosphoryl transfer enzymes involved in glucose metabolism

Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate. Phosph$\text{o}$ glucomutase and phosphoglycerate mutase are inhibited at the μM range. 2,3-Bisphosphoglycerate phosphatase is completely inhibited by 0.5 mM vanadate. 2,3-Bisphosphoglycerate synthase, hexokinase, phosphoglycerate kinase and fructose-1,6-P₂ phosphatase are partially inhibited by mM vanadate. Phosphofructokinase and pyruvate kinase are not affected. The glycolytic enzymes which mechanism does not involves phosphoryl transfer step are not affected by vanadate.     

Modification of the kinetics and regulatory properties of bovine hepatic fructose 1,6-diphosphatase with pyridoxal 5'-phosphate

Treatment of purified fructose 1,6-diphosphatase from bovine liver (which is maximally active at neutral pH) with pyridoxal 5'-phosphate produces changes in the spectral, catalytic, and allosteric properties of the enzyme. After modification the Michaelis constants for fructose-1,6-diphosphate and Mg²⁺ are increased, and inhibition by AMP is decreased. Substrate inhibition is decreased, but not abolished; the curve is merely shifted toward higher substrate concentration. Fructose-1, 6-diphosphate protects against the increases in the K_m for fructose-1, 6-diphosphate and the K_m for Mg²⁺, and against the changes in substrate inhibition, but not against the changes in AMP inhibition. AMP protects against the changes in AMP inhibition and the increase in the K_m for magnesium, but does not prevent the changes in substrate inhibition or the increase in the K_m for fructose-1, 6-diphosphate. The pH curves in the modified enzyme are altered at high, but not at low, substrate concentration.     

Effects of insulin,glucagon and dexamethasone on pyruvate kinase in cultured hepatocytes

Long-term (24–48 h) and short-term (10–30 min) regulation by hormones of hepatic pyruvate kinase activity was investigated in adult rat hepatocytes cultured under serum-free conditions. In the absence of hormones, pyruvate kinase total activity decreased to 83%, 67% and 39% of the initial level at 24, 48 and 72 h of culture. Insulin (100 nM) maintained total activity significantly above control levels throughout this period. In contrast, glucagon (100 nM) and dexamethasone (100 nM) accelerated the gradual decrease within 24 h (glucagon) or 48 h (dexamethasone) of culture. In these long-term experiments, activity at non-saturating concentrations of phosphoenolpyruvate was decreased by glucagon and dexamethasone but not directly modulated by insulin. However, insulin increased the cellular content of the pyruvate kinase activator fructose-1,6-diphosphate. In short-term experiments on cells cultured under serum- and hormone-free conditions for 48 h, both glucagon and dexamethasone independently caused a rapid, dose-dependent increase of the K_0.5 for phosphoenolpyruvate within 10 min, while V_max was not affected. Insulin inhibited this action of glucagon and dexamethasone and, in their absence, significantly increased substrate affinity for phosphoenolpyruvate within 30 min. Cellular fructose-1,6-diphosphate contents remained unchanged under these conditions. The data identify glucocorticoids and insulin - in addition to glucagon - as short-term regulators of the catalytic properties of pyruvate kinase. All three hormones are effective in the long-term control of total enzyme activity.     

Stringent control of glycolysis in Escherichia coli.

When $\text{Escherichia}\text{coli}\text{CP78}\text{(}\text{rel}{}^{\mathrm{+}}$) growing on glucose was starved for isoleucine by the addition of valine, the intracellular levels of fructose 6-phosphate, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were abruptly decreased to one-half, but those of glucose 6-phosphate and ATP remained constant. In contrast, this was not the case with CP79($\text{rel}{}^{\mathrm{?}}$). Chloramphenicol released the response observed in CP78. These results suggest that the glycolytic activity is also under the stringent control. Since only glucosephosphate isomerase[EC 5.3.1.9] was significantly inhibited by guanosine 5′-diphosphate 3′-diphosphate among several glycolytic enzymes tested, the enzyme might be responsible for the decrease observed in CP78.     

Crystallization of lactate dehydrogenase from Bacillus stearothermophilus

Crystals of lactate dehydrogenase from Bacillus stearothermophilus have been obtained in five different crystal morphologies belonging to at least two different space groups. Apo-lactate dehydrogenase can crystallize in space group P6₁22 or P6₅22 ($\text{a = 87}\text{A}\text{?}\text{and}\text{c = 358}\text{A}\text{?}$). A complex of lactate dehydrogenase with NADH and the effector fructose 1,6-diphosphate can crystallize in the same space group as the apoenzyme and in P6₃22 ($\text{a = 290}\text{A}\text{?}\text{, c = 146}\text{A}\text{?}$). Both forms are suitable for high resolution X-ray diffraction studies.     

Small-angle X-ray scattering of D-Ribulose-1,5-diphosphate carboxylase from Dasycladus clavaeformis roth (Ag.) in solution

D-Ribulose-1,5-diphosphate carboxylase from $\text{Dasycladus}$ was purified, and the gross dimensions were obtained by means of small-angle X-ray scattering measurements in solution. Dissolved single crystals of this enzyme (called “fraction I protein”) gave the same hydrodynamic parameters as the purified form. The molecular weight was found to be 535,000, and a radius of gyration of R_g = 45.5 Å was determined. The experimental scattering curves revealed a geometrical particle of D-Ribulose-1,5-diphosphate carboxylase with gross dimensions of that of a hollow sphere with outer radius of 56 Å and inner radius of 12 Å. Determinations of the diffusion coefficients lead to the conclusion that the enzyme has a spherical shape of almost uniform density.     

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.     

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.     

Chloroplast and Cytoplasmic Enzymes: IV. Pea Leaf Fructose 1,6-Diphosphate Aldolases

Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 μM for the chloroplast, 21 μM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 μM for the chloroplast enzyme, 18 μM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf.     

Metabolite concentrations in the liver of the adult and developing guinea pig and the control of glycolysis in vivo.

A range of metabolite concentrations have been determined in the liver of the adult and fetal guinea pig during the latter half of gestation. Adenine nucleotides showed little change during development of the fetal liver and the only major difference from the adult was a low ADP concentration. The hexose phosphates, particularly fructose 1,6-diphosphate, were higher and the triose phosphates in the glycolytic pathway after glyceraldehyde 3-phosphate were lower in the fetal liver. Cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratios were comparable in both adult and fetal livers as were cytosolic $\text{NADP}{}^{\mathrm{+}}\text{NADPH}$ ratios for the last 15–20 days of gestation. The metabolite concentrations have been used to indicate that glycolysis in the fetal guinea pig liver is controlled largely by hexokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate kinase.     

The extraction of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) from Escherichia coli using low pH reagents: a reevaluation.

It has been found that the most widely used method for the extraction of guanosine 5′-diphosphate, 3′-diphosphate (ppGpp) from $\text{E. coli}$ (1 M formic acid at 0°) results in its $\text{in vitro}$ degradation to ppGp and GDP. A comparison with several other extraction procedures indicated that this breakdown is due to the low pH of the reagents used during extraction. This degradation can largely be prevented by using a new extraction technique which involves freezing and thawing of the cells in the presence of lysozyme at a neutral pH followed by treatment with deoxycholate. With this method it is possible to recover from three to five times as much ppGpp from both unstarved and amino acid starved stringent strains of $\text{E. coli}$ as compared with the most widely used formic acid procedure. Consequently, it will be necessary to reevaluate the ppGpp values obtained from cells when formic acid or other low pH reagents were used during extraction.     

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.