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At least 307 records · Page 17Linked to original sources
PubMed1976

The control of pyruvate kinase of Escherichia coli. Binding of substrate and allosteric effectors to the enzyme activated by fructose 1,6-bisphosphate.

The binding of various regulatory ligands and substrates to the fructose bisphosphate activated pyruvate kinase from Escherichia coli has been studied at equilibrium. The allosteric activator, fructose bisphosphate, and the substrate phosphoenolypyruvate bind in a cooperative manner to the enzyme. There is one site for each of these ligands per monomer. In the presence of fructose bisphosphate the binding of phosphoenolpyruvate follows an absorption isotherm, i.e., all homotropic interactions of the substrate are lost. In reciprocal experiments, however, both phosphoenolpyruvate and KCl are required in order to facilitate binding of the activator. The allosteric inhibitors of pyruvate kinase, ATP, succinyl-CoA, and GTP compete on the enzyme surface with the binding of the activator, fructose bisphosphate, Inhibitor pairs such as ATP and succinyl-CoA together bring about not cooperative but only additive inhibition of the binding of the activator. The nucleotide substrate GDP and the allosteric inhibitor GTP have in contrast to the activator two seemingly noninteracting sites on each monomer. In the saturating presence of fructose bisphosphate, however, binding of GDP and possibly also of GTP occurs at only one site on each monomer. Magnesium ions inhibit binding of GDP and GTP. KCl which is an activator of the enzyme along with its analogues, such as ammonia, thallium, rubidium, etc., enhances the binding of phosphoenolpyruvate but not of the nucleotides or fructose bisphosphate. The data are analyzed on the basis of a two-site model, where the substrate and fructose bisphosphate bind to one conformation and the inhibitors to the other.

Allosteric Regulation↗
PubMed1976

Fructose 1,6-bisphosphate: isomeric composition, kinetics, and substrate specificity for the aldolases.

13C NMR shows fructose 6-phosphate and fructose 1,6-bisphosphate to contain respectively 4.1 and 2.0% keto isomer at room temperature. The lower value for fructose 1,6-bisphosphate can be attributed to the electron-withdrawing effect of the C-1 phosphate. Measurements of the ring-opening rates of the alpha and beta anomers of fructose, 1,6-bisphosphate by an NMR line-broadening technique show them to be about 8 and 35 S-1, respectively, at pH 7.2, and 25degreesC. The value for the predominant beta anomer is threefold greater than the turnover rate of muscle aldolase so that, if the kinetic properties of the keto form were favorable, the reaction could proceed entirely through the keto form in solution. The kinetic properties of a fructose 1,6-bisphosphate(keto) analogue, 5-deoxyfructose, 1,6-bisphosphate, in the muscle aldolase reaction are more favorable (Vmax = 2.6, Km = 0.11 X 10(-6) M) than those of fructose 1,6-bisphosphate total (Vmax = 1, Km = 2.3 X 10(-6)M), giving a value of Vmax/Km that is 56 times greater for the 5-deoxy analogue. At the 2.0% concentration of the keto form this is sufficient to account for the steady-state rate and requires that the beta form, present at 40 times greater concentration, contributes little to the cleavage rate. With yeast aldolase the cleavage rate can be explained by the rapid spontaneous ring opening and reaction of the keto form with the enzyme. In view of the high rate of ring opening and the excellent properties of the keto form, previous rapid kinetic studies favoring action of cyclic forms may require reevaluation.

Fructose-Bisphosphate Aldolase↗
PubMed1975

An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase.

Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.

Animals↗
PubMed1975

Hysteretic characteristic of adenine phosphoribosyltransferase.

Preassay-incubation of the highly purified human erythrocyte adenine phosphoribosyltransferase (EC 2.4.2.7) (AMP pyrophosphorylase) with one of its substrates, 5-phosphoribosyl 1-pyrophosphate (PRibPP), changes the apparent V max value of the enzyme reaction. The extent of inhibition by preassay-incubation with an inhibitor, fructose 1,6-diphosphate (FDP), or a destabilizer, hypoxanthine (Hx), is found not to be proportional to the amount of the inhibitor present. The maximum inhibition achieved by preassay-incubation was about 40%. The PRibPP, FDP, and Hx induced changes in AMP pyrophosphorylase do not require the presence of divalent ions. The inhibtion of AMP pyrophosphorylase produced by preincubation with Hx was prevented when PRibPP was added to the preassay-incubation system. However, the preassay-incubation effect of FDP was only partially diminished under the same conditions. Contrary to the PRibPP-bound AMP pyrophosphorylase, the adenine-bound enzyme was found to be more heat labile than the unbound enzyme. Similar thermal instability was also observed with FDP- and Hx-bound enzyme. Our experimental results indicate that a conformational change of AMP pyrophosphorylase induced by the binding of metabolites is a slow process as compared to the overall catalytic reaction. This hysteretic characteristic of AMP pyrophosphorylase may be one of the regulatory mechanisms in purine intermediary metabolism.

Adenine Phosphoribosyltransferase↗
PubMed2005

Hexitol nucleic acid-containing aptamers are efficient ligands of HIV-1 TAR RNA.

The transactivation responsive element (TAR) plays a crucial role in the transcription of the HIV-1 genome upon specific binding of the viral protein Tat and cellular proteins. We have previously identified a RNA hairpin aptamer forming a stable and specific kissing complex with TAR RNA (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). We chemically modified this aptamer with hexitol nucleic acid (HNA) residues. We demonstrate that a fully HNA-modified aptamer is a poor ligand but, in contrast, mixmers containing both HNA and unmodified RNA nucleotides display interesting properties. Two HNA-RNA mixmers bind to TAR with an equilibrium dissociation constant in the low-nanomolar range and show a reduced nuclease sensitivity. In addition, they show a moderate dependence on magnesium ions for binding to TAR. These HNA-RNA mixmers are able to inhibit transactivation of transcription in an in vitro assay.

Base Sequence↗
PubMed1989

Fructose-1,6-bisphosphate protects astrocytes from hypoxic damage.

To determine the effects of glucose and fructose-1,6-bisphosphate (FDP) on hypoxic cell damage, primary cultures of astrocytes were incubated for 18 h in an air-tight chamber that had been flushed with 95% N2/5% CO2 for 15 min before it was sealed. Cultures containing 7.5 mM glucose without FDP or FDP without glucose showed evidence of significant cell injury after 18 h of hypoxia (increased lactate dehydrogenase content in the culture medium; cell edema and disruption by phase-contrast microscopy). Cultures exposed to glucose + FDP had normal lactate dehydrogenase concentrations and appeared normal microscopically. Maximal protection of hypoxic cells occurred at 6.0 mM FDP. Lactate concentrations of the culture medium of hypoxic cells increased 2.5 times above normoxic control values when glucose was present, but neither FDP alone nor glucose + FDP caused the lactate concentrations to increase further. This implies that anaerobic glycolysis was not increased by adding FDP to the medium. Cell volumes (water space) measured with [14C]-3-0-methyl-D-glucose were normal with glucose + FDP in the culture medium of hypoxic cells but were significantly larger than normal when glucose alone was present. Increases in cell volume paralleled changes in lactate dehydrogenase in the culture medium. Uptake of [14C]FDP occurred rapidly in normoxic cells and was maximal after 5 min of incubation. The data indicate that the presence of glucose + FDP in the culture medium protects primary cultures of hypoxic astrocytes from cell damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1973

Kinetic studies on the regulation of rabbit liver pyruvate kinase.

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).

Adenosine Diphosphate↗
PubMed1973

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.

Allosteric Regulation↗
PubMed1973

Lack of temperature-sensitivity of rat liver pyruvate kinase.

Rat liver pyruvate kinase, prepared by Sephadex treatment of a 10(5)g supernatant in phosphate buffer, is quite stable and gives reproducible results when a variety of parameters are altered in the enzyme assay. Incubation of this preparation at 25 degrees C or 0-2 degrees C has no effect on the activation by fructose 1,6-diphosphate or inhibition by ATP or alanine.

Adenosine Triphosphate↗
PubMed1974

Subunit interactions in hybrids of native, carboxypeptidase-treated and citraconylated rabbit muscle aldolase.

1. The kinetic properties of hybrids of native (or carboxypeptidase-treated) and citraconylated rabbit muscle aldolase are compared with those of equivalent mixtures of the parental enzymes. 2. In the hybrids, the native subunits function slightly less well than in the homotetramer, but the citraconylated subunits have enhanced activity. 3. Subunits of carboxypeptidase-treated aldolase behave essentially as expected in a hybrid environment, but the citraconylated subunits do not show the same enhancement of activity found in the hybrids of native and citraconylated enzyme. The apparent affinity for fructose 1,6-diphosphate of the citraconylated subunits in hybrids of carboxypeptidase-treated and citraconylated aldolase is increased. 4. These results are interpreted in terms of a substrate-induced conformational difference between native and carboxypeptidase-treated aldolase. 5. This conformational change can take place within a single native subunit in the hybrids and does not require a similar conformational change to occur simultaneously in the other three subunits.

Acylation↗
PubMed1977

Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex.

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.

Animals↗
PubMed1978

Maximum activities and effects of fructose bisphosphate on pyruvate kinase from muscles of vertebrates and invertebrates in relation to the control of glycolysis.

1. Comparison of the maximum activities of pyruvate kinase with those of phosphofructokinase in a large number of muscles from invertebrates and vertebrates indicates that, in general, in any individual muscle, the activity of pyruvate kinase is only severalfold higher than that of phosphofructokinase. This is consistent with the suggestion, based on mass-action ratio data, that the pyruvate kinase reaction is non-equilibrium in muscle. However, the range of activities of pyruvate kinase in these muscles is considerably larger than that of phosphofructokinase. This difference almost disappears if the enzyme activities from muscles that are known to possess an anaerobic ;succinate pathway' are excluded. It is suggested that, in these muscles, phosphofructokinase provides glycolytic residues for both pyruvate kinase (i.e. glycolysis) and phosphoenolpyruvate carboxykinase (i.e. the succinate pathway). This is supported by a negative correlation between the activity ratio, pyruvate kinase/phosphofructokinase, and the activities of nucleoside diphosphokinase in these muscles, since high activities of nucleoside diphosphokinase are considered to indicate the presence of the succinate pathway. 2. The effect of fructose bisphosphate on the activities of pyruvate kinase from many different muscles was studied. The stimulatory effect of fructose bisphosphate appears to be lost whenever an efficient system for supply of oxygen to the muscles is developed (e.g. insects, squids, birds and mammals). This suggests that activation of pyruvate kinase is important in the co-ordinated regulation of glycolysis in anaerobic or hypoxic conditions, when the change in glycolytic flux during the transition from rest to activity needs to be large in order to provide sufficient energy for the contractile activity. However, lack of this effect in the anaerobic muscles of the birds and mammals suggests that another metabolic control may exist for avian and mammalian pyruvate kinase in these muscles.

Animals↗
PubMed1980

A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas.

1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.

Adenosine Diphosphate↗