Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2-Keto-3-deoxygluconate-6-P exists as an euqilibrium of three forms at 25 degrees measurable by 13C NMR: alpha-furanose anomer (41%), beta-furanose anomer (50%), and open chain keto (9%). The three forms are interconverted rapidly (greater than 0.5 s-1) so that the unidirectional rates of furanose ring opening and closing can be quantitated by an NMR line broadening method. The 2-keto-3-deoxygluconate aldolase is specific for only one of these forms, the open chain keto form. The rates for ring opening calculated from the rapid kinetic enzyme system compare closely with those obtained with the NMR method.
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PMID:Specificity of 2-keto-3-deoxygluconate-6-P aldolase for open chain form of 2-keto-3-deoxygluconate-6-P. 1 4

Anaerobic glycolysis in Saccharomyces cerevisiae has been studied by 13C NMR at 90.5 MHz. [1-13c]Glucose and [6-13C]glucose were fed to suspensions of yeast cells. Time courses for concentration changes of the starting material, of courses for concentration changes of the starting material, of the intermediate fructose 1,6-bisphosphate (Fru-P2), and of the end products, ethanol and glycerol, have been followed with 1-min time resolution. The glucose uptake was well fitted by a Michaelis-Menten model, assuming competition of alpha- and beta-glucose for the same site. The Km for the uptake was found to be 10 mM for beta-glucose and 5 mM for alpha-glucose. The concentration of Fru-P2 showed an initial oscillation before it reached a co,stant level. The 13C label, introduced only as [-13C]- or [6-13C]glucose, was observed in Fru-P2 in both the C1 and C6 positions, simultaneously. From the relative intensities of the C1 Fru-P2 and C6 Fru-P2 peaks in the presence of [1-13C]- and [6-13C]glucose, in vivo kinetic information was obtained about the aldolase-triosephosphate isomerase triangle. We found that under the conditions of these experiments the ratios of backward to forward velocities through aldolase and triosephosphate isomerase were 0.9 +/- 0.1 and 0.8 +/- 1, respectively, indicating they were close to equilibrium.
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PMID:13C nuclear magnetic resonance studies of anaerobic glycolysis in suspensions of yeast cells. 4 10

Fructose 6-sulfate was synthesized by direct sulfurylation of fructose and was isolated by two selective steps: (a) conversion of the 6-sulfuryl ester to fructose 1-phosphate-6-sulfate with phosphofructokinase; (b) conversion of fructose 1-phosphate-6-sulfate to fructose 6-sulfate by fructose-1,6-diphosphatase. Utilizing crystalline sheep heart phosphofructokinase, kinetic studies with the alternative substrate were carried out at pH 8.2 which is optimal for nonallosteric kinetics. The data are consistent with an ordered addition of the two substrates with the first, MgATP, being at thermodynamic equilibrium. The Vmax and Km obtained with fructose 6-sulfate were 0.03- and 100-fold, respectively, that obtained with the natural substrate. The study suggests that the divalent phosphoryl moiety is intimately involved in the active site conformation. Identification of the product of the reaction, fructose 1-phosphate-6-sulfate, was confirmed through studies with aldolase, fructose-1,6-diphosphatase, and by 31P NMR. The utilization of fructose 6-sulfate as a substrate by yeast glucose-6-phosphate isomerase could not be demonstrated.
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PMID:Studies on heart phosphofructokinase. Use of fructose 6-sulfate as an alternative substrate to study the mechanism of action and active site specificity. 13 39

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.
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PMID:Fructose 1,6-bisphosphate: isomeric composition, kinetics, and substrate specificity for the aldolases. 77 19

Rabbit muscle aldolase catalyzes the exchange with solvent of all three methyl hydrogens of hydroxyacetone phosphate. Under saturating conditions, rates of the following processes have been measured: deuteration of hydroxyacetone phosphate in 2H2O (by an NMR method), tritiation of hydroxyacetone phosphate in H2O and 2H2O, and detritiation of tritiated hydroxyacetone phosphate in H2O and 2H2O. It is clear from these measurements (1) that there is no primary kinetic isotope effect and hence that hydrogen abstraction is not rate determining to the exchange and (2) that only one (as the closest integer) methyl hydrogen exchanges per turnover. The argument is made that these observations are mutually exclusive in terms of the accepted aldolase mechanism in the absence of further restrictions imposed by the enzyme. Possible restrictions are discussed.
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PMID:Rabbit muscle aldolase catalyzed proton exchange of hydroxyacetone phosphate with solvent. 91 52

In order to elucidate the effects of amphotericin B (AMB) on the glycolytic pathway, the metabolism of [1-13C]glucose in glucose-grown repressed Saccharomyces cerevisiae was studied. The cells were aerobically suspended in pyrophosphate solutions of high potassium concentration with or without 10(-6) M amphotericin B and measurements were made using 1H-, 13C-NMR spectroscopy and biochemical methods. The results were compared with those obtained under the same experimental conditions but in a medium rich in sodium salts containing the same antibiotic concentration. In general the presence of 10(-6) M AMB reduces the glucose consumption and the ethanol production while favouring the glycerol and trehalose formation. These effects are greatly reduced when a high K+ concentration was used. The AMB effects on the glucose consumption and the production of ethanol, glycerol and trehalose, observed in a suspension rich in Na+, can be fairly well explained by the leakage of K+ through AMB membrane channels. This outflux induces a substantial decrease in the activity of some K(+)-dependent enzymes, such as aldolase, phosphofructokinase and pyruvate kinase. The intensities of the glutamate C2 and C4 signals are higher with a suspension rich in Na+ than with a suspension rich in K+, suggesting that the Krebs cycle operates more effectively in a solution rich in Na+. In the absence of AMB, the passive diffusion of glycerol through the cell membrane is relatively slow and apparently depends on the ionic external medium: it is more efficient in solutions with a high K+ than with a high Na+ concentration. In the presence of 10(-6) M AMB, the glycerol C1,3 resonance drastically decreases at 20 min and then disappears in the noise. This rapid disappearance suggests that glycerol can easily pass through the pores arising from the interaction of AMB with the membrane sterols. However, the rate of pore formation is slow, independent of the external medium (Na+ or K+) and this process is not completed within 20 min.
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PMID:Comparative study of the effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae in K(+)- and Na(+)-rich media. 132 8

NMR spectroscopy showed fructose-1,6-bisphosphate aldolase from rabbit muscle accepts as substrates, in lieu of glyceraldehyde 3-phosphate, the oxoaldehydes methylglyoxal and phenylglyoxal but not hydroxymethylglyoxal. The enzyme catalyzed an aldol condensation between the oxoaldehyde and dihydroxyacetone phosphate to form a monophosphorylated diketone and was inactivated in the process. Circumvention of this reaction, by metabolism of oxoaldehydes to hydroxy acids, may be a metabolic role for the glyoxalase enzyme system. Transketolase and transaldolase were found not to accept oxoaldehydes as substrates in place of glyceraldehyde 3-phosphate.
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PMID:Aldolase-catalyzed diketone phosphate formation from oxoaldehydes. NMR studies and metabolic significance. 157 6

Reductive, nonreductive, and photolytic interactions of vanadate with fructose-1,6-bisphosphate aldolase were examined and used to explore the interactions of oxoanions with aldolase. Aldolase is known to interact strongly with oxoanions at low ionic strength and weakly at higher ionic strength. Oxoanions inhibit aldolase competitively with respect to fructose 1,6-bisphosphate although the location of the oxoanion binding site on aldolase remains elusive. In this work, the interaction of aldolase with a series of oxoanions was compared at ionic strength approaching physiologic levels. The size and shape of the anion were important for the effective binding to aldolase, and no significant increase in affinity for aldolase was observed by the addition of alkyl groups to the oxoanions. Vanadate competitively inhibits aldolase in a manner analogous to the other oxoanions. Since vanadate solutions contain a mixture of vanadate oxoanions, the nature of the inhibition was determined using a combination of enzyme kinetics and 51V NMR spectroscopy. Aldolase contains a significant number of thiol functionalities, and as expected, vanadate undergoes redox chemistry with them, generating an irreversibly inhibited aldolase. This oxidative chemistry was attributed to the vanadate tetramer, whereas vanadate dimer was a reversible inhibitor. Vanadate monomer does not significantly interact with aldolase reversibly or irreversibly. Vanadyl cation has the lowest inhibition constant under these high ionic strength conditions. Using Yonetani-Theorell analysis, it appears that phosphate, pyrophosphate, and sulfate bind to the same site on aldolase, whereas vanadate, arsenate, and molybdate bind to another site. UV light-induced photocleavage of aldolase by vanadate was examined, and the loss of aldolase activity was correlated with cleavage of the aldolase subunit. Further studies using vanadium as a probe should reveal details on the location of the vanadate and vanadyl cation binding sites. This study suggests several sites on aldolase will accommodate oxoanions, and one of these sites also accommodates vanadyl cation.
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PMID:Interaction of rabbit muscle aldolase at high ionic strengths with vanadate and other oxoanions. 163 17

Glucose carbon recycling, glucose production and glucose turnover in glycogen storage disease type I and type II patients and control subjects were determined by a novel approach--mass isotopomer analysis of plasma 13C glucose. Changes in the isotopomer distribution of plasma 13C glucose were found only in glycogen storage disease type III patients and control subjects. Glucose carbon recycling parameters were also derived from 13C NMR spectra of plasma glucose C-1 splitting pattern. Our results eliminate a mechanism for glucose production in glycogen storage disease type I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity is in agreement with our results. A quantitative determination of the metabolic pathways of fructose conversion to glucose in normal children, and in children with disorders of fructose metabolism was derived from 13C NMR measurement of plasma 13C glucose isotopomer populations following [U-13C]fructose administration. A direct pathway from fructose, bypassing fructose-1-phosphate aldolase, to fructose-1,6-diphosphate in controls and hereditary fructose intolerant children (47% and 27%, respectively) was identified. In children with fructose-1,6-diphosphatase deficiency, only the gluconeogenic substrates were 13C labelled but no synthesis of glucose from [U-13C]fructose occurred. The significantly lower (by 68%) conversion of fructose to glucose in hereditary fructose intolerance, as compared to control subjects, and non-conversion in fructose-1,6-diphosphatase deficient subjects after [U-13C]fructose (approximately 20 mg/kg) administration can serve as the basis of a safe diagnostic test for patients suspected of inborn errors of fructose metabolism and other defects involving gluconeogenesis.
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PMID:Inherited disorders of carbohydrate metabolism in children studied by 13C-labelled precursors, NMR and GC-MS. 212 13

An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U-13C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13C NMR spectra of plasma glucose. The conversion parameters in the control and HFI children were estimated on the basis of doublet/singlet values of the plasma beta-glucose C-1 splitting pattern as a function of the rate of fructose infusion (0.26-0.5 mg/kg per min). Significantly lower values (approximately 3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13C NMR measurement of plasma [13C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13C atoms at glucose C-4 (13C3-13C4-13C5) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only approximately 50% of the total amount of hepatic fructose conversion to glucose. It is suggested that phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate by 1-phosphofructokinase occurs in human liver (and intestine) when fructose is administered nasogastrically; 47% and 27% of the total fructose conversion to glucose in controls and in HFI children, respectively, takes place by way of this pathway. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [13C]glucose formation from a "trace" amount (approximately 20 mg/kg) of [U-13C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.
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PMID:Determination of fructose metabolic pathways in normal and fructose-intolerant children: a 13C NMR study using [U-13C]fructose. 237 Dec 80


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