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.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Activation of ribulose 1,5-diphosphate carboxylase by nicotinamide adenine dinucleotide phosphate and other chloroplast metabolites

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 10 mm MgCl₂ and 1 mm bicarbonate in the absence of ribulose 1,5-diphosphate, can be further activated about 170% with 0.5 mm NADPH present in the preincubation mixture. NADP⁺, NADH, and NAD⁺ are ineffective. The activation by NADPH is comparable to that previously seen with 0.05 to 0.10 mm 6-phosphogluconate in that these specific preincubation conditions are required, but the effects of NADPH and 6-phosphogluconate are not additive. Moreover, where higher concentrations of 6-phosphogluconate inhibited the enzyme, higher concentrations of NADPH give a greater activation, saturating at about 1 mm and 200%. Under the specified conditions of preincubation, fructose 1,6-diphosphate has an activation curve similar to that of 6-phosphogluconate, peaking at 0.1 mm and 70%. Above this level, activation decreases, and inhibition is seen at still higher concentrations. Other metabolites tested produced smaller or no effects on the enzyme activity assayed under these conditions. When either reduced NADP or 6-phosphogluconate are present in the preincubation mixture, it becomes possible to determine the Km for bicarbonate using a Lineweaver-Burk plot, and the Km for bicarbonate under these conditions is 2.8 mm, corresponding to 0.3% CO₂ at pH 7.8 and 25 C.     

Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis

1. Phosphofructokinase from rat liver has been partially purified by ammonium sulphate precipitation so as to remove enzymes that interfere in one assay for phosphofructokinase. The properties of this enzyme were found to be similar to those of the same enzyme from other tissues (e.g. cardiac muscle, skeletal muscle and brain) that were previously investigated by other workers. 2. Low concentrations of ATP inhibited phosphofructokinase activity by decreasing the affinity of the enzyme for the other substrate, fructose 6-phosphate. Citrate, and other intermediates of the tricarboxylic acid cycle, also inhibited the activity of phosphofructokinase. 3. This inhibition was relieved by either AMP or fructose 1,6-diphosphate; however, higher concentrations of ATP decreased and finally removed the effect of these activators. 4. Ammonium sulphate protected the enzyme from inactivation, and increased the activity by relieving the inhibition due to ATP. The latter effect was similar to that of AMP. 5. Phosphofructokinase was found in the same cellular compartment as fructose 1,6-diphosphatase, namely the soluble cytoplasm. 6. The properties of phosphofructokinase and fructose 1,6-diphosphatase are compared and a theory is proposed that affords dual control of both enzymes in the liver. The relation of this to the control of glycolysis and gluconeogenesis is discussed.     

Regulatory characteristics of a fructose bisphosphatase from the blue-green bacterium Anacystis nidulans.

A specific fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from the obligately autotrophic blue-green bacterium Anacystis nidulans. It was most active at pH 8.0. The K_m for fructose 1,6-bisphosphate was 0.088 mm at pH 8.0 and 0.105 mm at pH 7.0; the K_m for MgCl₂ was 0.95 mm at pH 8.0. Activity at netural pH was particularly sensitive to the MgCl₂ concentration. AMP was an allosteric inhibitor, 50% inhibition being exerted by 0.058 mm AMP at pH 7.0 and 0.085 mm AMP at pH 8.O. The way in which changes in intracellular pH and the concentrations of Mg²⁺ and AMP might influence the activity of the enzyme in the Calvin cycle, the oxidative pentose phosphate pathway and in glycolysis and gluconeogenesis is discussed.     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts

Millimolar concentrations of Mg²⁺ inhibited CO₂-dependent O₂ evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O₂ evolution by Mg²⁺ and reduced the Mg²⁺ inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O₂ evolution by Mg²⁺ whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg²⁺ inhibition of O₂ evolution shortened the lag phase of CO₂-dependent O₂ evolution in the absence of M²⁺. Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O₂ evolution and the inhibition of O₂ evolution by Mg²⁺. The results suggested that Mg²⁺ inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.     

The effect of Zn2+ on the inhibition of Fru-P2ase by AMP.

In the absence of chelating agents, the sensitivity of rabbit liver fructose 1,6-bisphosphatase to inhibition by AMP is increased by approximately 10-fold, apparently related to the presence of bound Zn²⁺. At pH 7.5, 10 μM AMP causes virtually complete inhibition and nearly full activity is restored by the addition of chelating agents such as EDTA or histidine. These properties provide a mechanism for the induction of Fru-P₂ase activity under gluconeogenic conditions.     

Nickel in plants: I. Uptake kinetics using intact soybean seedlings

The absorption of Ni²⁺ by 21-day-old soybean plants (Glycine max cv. Williams) was investigated with respect to its concentration dependence, transport kinetics, and interactions with various nutrient cations. Nickel absorption, measured as a function of concentration (0.02 to 100 μm), demonstrated the presence of multiple absorption isotherms. Each of the three isotherms conforms to Michaelis-Menten kinetics; kinetic constants are reported for uptake by the intact plant and for transfer from root to shoot tissues. The absorption of Ni²⁺ by the intact plant and its transfer from root to shoot were inhibited by the presence of Cu²⁺, Zn²⁺, Fe²⁺, and Co²⁺. Competition kinetic studies showed Cu²⁺ and Zn²⁺ to inhibit Ni²⁺ absorption competitively, suggesting that Ni²⁺, Cu²⁺, and Zn²⁺ are absorbed using the same carrier site. Calculated K_m and K_i constants for Ni²⁺ in the presence and absence of Cu²⁺ were 6.1 and 9.2 μm, respectively, whereas K_m and K_i constants were calculated to be 6.7 and 24.4 μm, respectively, for Ni²⁺ in the presence and absence of Zn²⁺. The mechanism of inhibition of Ni²⁺ in the presence of Fe²⁺ and Co²⁺ was not resolved by classical kinetic relationships.     

Selective modification of rabbit liver fructose bisphosphatase

Chemical modification of rabbit liver fructose 1,6-bisphosphatase by 5,5′-dithiobis-(2-nitrobenzoic acid) results in thiolation of four highly reactive sulfhydryl groups and a diminished sensitivity to AMP inhibition but not loss of enzyme activity. Ethoxyformylation of the histidine groups of fructose 1,6-bisphosphatase does not result in a sharp loss of activity until at least 4 or 5 of the 13 residues have reacted. Exhaustive formylation does abolish the enzyme's activity. These four most reactive sulfhydryl groups and the one or two least easily modified histidine moieties (those responsible for activity) can be protected against modification by fructose-1,6-P₂ and to a lesser extent by fructose-6-P. The binding of fructose-1,6-P₂ to fructose 1,6-bisphosphatase, however, depends on the presence of structural metal ion since EDTA which removes all endogenous Zn²⁺ from the protein prevents binding of fructose-1, 6-P₂ to the enzyme.     

Some Properties of Partially Purified Pyruvate Kinase from Euglena gracilis Klebs var. bacillaris

A method of purification of pyruvate kinase (EC 2.7.1.40) from light-grown Euglena gracilis var. bacillaris was developed which yielded an enzyme preparation purified 115-fold over crude extracts. During organelle formation, levels of pyruvate kinase in extracts prepared from cells engaged in light-induced chloroplast development do not change significantly. The enzyme has a molecular weight of approximately 240,000 and a requirement for both K⁺ and Mg²⁺. Fructose 1,6-diphosphate activates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentration of ADP is limiting. ATP, citrate, and Ca²⁺ are inhibitors of the enzyme and inhibit the fructose 1,6-diphosphate stimulation of the enzyme activity. ATP inhibition is only partially reversed by high concentrations of fructose 1,6-diphosphate. Further reversal of inhibition can be achieved by dialysis. Ca²⁺-dependent inhibition can be reversed by a chelating agent but not by increased concentrations of Mg²⁺.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Reversal of glycolysis in yeast.

When a buffered, aerobic suspension of ethanol-grown cells of Saccharomyces cerevisiae is treated with ethanol, a rapid flux of metabolism is observed from endogenous phosphoenolpyruvate to hexose monophosphates. Intracellular concentrations of phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate record a monotonic drop, while those of triose phosphates and fructose 1,6-diphosphate fall after an early rise; fructose 6-phosphate, mannose 6-phosphate, and glucose 6-phosphate levels rise to a plateau. Prior growth on glucose extinguishes fructose 1,6-diphosphatase activity and completely arrests the rise of the hexose monophosphates. By using mutants blocked at a number of glycolytic steps it has been concluded that the metabolic flow takes place along the Embden-Meyerhof pathway in the reverse direction bypassing pyruvate kinase and fructose 6-phosphate kinase. Ethanol acts as a trigger by supplying NADH at the glyceraldehyde 3-phosphate dehydrogenase step. The rate of the reversal in the span phosphoenolpyruvate to fructose 1,6-diphosphate approaches 40 μ mol of 3-carbon units per minute per gram of wet cells. The in vivo activity of fructose 1,6-diphosphatase is nearly a quarter of this rate.     

Hymenolepis diminuta: Partial characterization of the membrane-bound and solubilized alkaline phosphohydrolase activities of the isolated brush border plasma membrane

The membrane-bound and solubilized (using Triton ×-100 or sodium dodecyl sulfate (SDS)) alkaline phosphohydrolase (APase) activities of the isolated brush border membrane of Hymenolepis diminuta require a divalent cation for maximum activity. Highest rates of substrate (p-nitrophenyl phosphate) hydrolysis are obtained with low concentrations of Mg²⁺ (1 mM), although low concentrations of Mn²⁺, Ca²⁺, or Zn²⁺ will also partially satisfy this requirement; higher concentrations of Mg²⁺ and Mn²⁺, and other divalent cations (Cu²⁺, Fe²⁺, and Pb²⁺), inhibit the membrane-bound APase activity. Solubilization of the membrane-bound enzyme in either Triton or SDS results in an increase in specific activity and K_m, but has little effect on thermal stability of the APase activity. Phosphate, pyrophosphate, adenosine 5′-triphosphate, adenosine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate inhibit substrate hydrolysis, and the relative affinities of these inhibitors for the APase enzyme are altered only slightly upon solubilization. Graphic analyses of data from inhibitor studies indicate that all eight inhibitors will inhibit membrane-bound and solubilized APase activities 100% at high inhibitonsubstrate ratios. Molybdate, F^?, 2-mercaptoethanol, cysteine, and p-chloromercuribenzoate inhibit membrane-bound APase activity. Inhibitor data indicate that if more than one enzyme is responsible for the APase activity of the brush border membrane of H. diminuta, the enzymes cannot be differentiated on the basis of substrate specificity.     

Ferrochelatase of Rhodopseudomonas spheroides

Extracts of Rhodopseudomonas spheroides contain two ferrochelatases: one is soluble and forms metalloporphyrins from deuteroporphyrin and haematoporphyrin; the other is particulate and forms metalloporphyrins from protoporphyrin, mesoporphyrin, deuteroporphyrin and haematoporphyrin. Neither enzyme incorporates Mg²⁺ into porphyrins or Fe²⁺ into porphyrin cytochrome c. By using the particulate enzyme, plots of 1/v versus 1/s when one substrate was varied and the other kept constant showed that neither substrate affected the K_m of the other. The suggested sequential mechanism for the reaction is supported by derivative plots of slopes and intercepts. The K_m for deuteroporphyrin was 21.3μm and that for Co²⁺ was 6.13μm. The enzyme incorporated Co²⁺, Fe²⁺, Zn²⁺, Ni²⁺ and Mn²⁺; Cd²⁺ was not incorporated and was an inhibitor, competitive with respect to Co²⁺, non-competitive with respect to deuteroporphyrin. The K_i for Cd²⁺ was 0.73μm. Ferrochelatase was inhibited by protohaem, non-competitively with respect to Co²⁺ or with respect to deuteroporphyrin. Inhibition by magnesium protoporphyrin was non-competitive with respect to deuteroporphyrin, uncompetitive with respect to Co²⁺. The inhibitory concentrations of the metalloporphyrins are lower than those required for the inhibition of δ-aminolaevulate synthetase by protohaem. Fe²⁺ is not incorporated aerobically into porphyrins unless an electron donor, succinate or NADH, is supplied; the low aerobic rate of metalloporphyrin synthesis obtained is insensitive to rotenone and antimycin. The rate of Fe³⁺ incorporation increases as anaerobic conditions are achieved.     

Purification and Properties of Fructokinase from Developing Tubers of Potato (Solanum tuberosum L.)

Fructokinase has been purified from developing potato (Solanum tuberosum L.) tubers by a combination of hydrophobic interaction, affinity chromatography, and gel filtration. The protein has a native molecular mass of approximately 70 kD but is apparently a dimer. Ion-exchange chromatography and two-dimensional western blots resolved three major fructokinases, designated FK-I, FK-II, and FK-III in order of their elution from a Mono-Q column. Fructokinase activity proved labile when proteins were purified in the absence of fructose. Kinetically, FKs I, II, and III all have broad pH optima with peaks at about pH 8.5. The enzymes have a high specificity for fructose (K_m values ranging from 0.041 to 0.128 mm), and can utilize a range of nucleoside triphosphates. Unlike FKs I and II, FK-III is not inhibited by fructose concentrations in excess of 1 mm. MgADP inhibited activity of the three FKs (between 68 and 75% inhibition at 1.0 mm), whereas fructose 6-P caused inhibition at concentrations of 10 mm. There were no regulatory effects observed with a range of other metabolites. K⁺ (10 mm) activated FK-I by 4-fold and FKs II and III by only about 50%.     

Effect of substrate and AMP on the reversal of Zn2+ inhibition of turkey liver fructose-1,6-bidphosphatase by chelators

The ability of chelators to reverse Zn²⁺ inhibition of turkey liver fructose-1,6-bisphosphatase decreases greatly if substrate is first bound to the enzyme. If AMP is also present, chelators become almost completely incapable of reversing Zn²⁺ inhibition when added to the enzyme after substrate. These observations indicate that the prior binding of substrate to this fructose-1,6-bisphosphatase hinders the removal of Zn²⁺ from the inhibitory sites of the enzyme by chelators, especially when AMP is also present. We have also found that the initial rates of the Zn²⁺-inhibited enzyme activity show a peculiar nonlinearity and the inhibitory effects of Zn²⁺ and AMP are synergistic.     

Fructose 1,6-diphosphatase in striated muscle

1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 μmoles of substrate were split/g. fresh wt./hr. at 22°. 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0·1 mm, by AMP and by trace quantities of Zn²⁺, Fe²⁺ and Fe³⁺; it is `activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C₃ compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C₃ intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. α-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of α-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.