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:2.7.1.1 (hexokinase)
5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reciprocal effects of 2-deoxy-D-glucose (dGlc) and glucose (Glc) on the aerobic metabolism of Glc and dGlc in Glc-grown repressed Saccharomyces cerevisiae were studied at 30 degrees C in a standard pyrophosphate medium containing about 4.5 x 10(7) cells/ml. 1H-, 13C-NMR and biochemical techniques were used for quantitative evaluation of Glc and dGlc metabolites. The detection of intracellular dGlc and the determination of the intracellular dGlc6P/dGlc ratio were realised using [1-13C]dGlc and 13C-NMR spectroscopy. The rates of Glc consumption in the absence and in the presence of 5 mM dGlc and 20 mM dGlc were 29 +/- 0.03 mM/min (n = 3), 16 +/- 0.02 mM/min (n = 3), and 0.08 +/- 0.01 mM/min (n = 3), respectively. This means that the Glc consumption is reduced about 41% and 70% in the presence of 5 mM and 20 mM dGlc, respectively. When dGlc is the unique carbon source, only alpha and beta anomers of dGlc6P were formed. Their quantities are equivalent and reach the maximum values within 1 h of incubation and then decrease gradually. By contrast, Glc favours the consumption of dGlc and the synthesis of dGlc6P, dideoxy-trehalose (dGlc-dGlc), deoxy-trehalose (dGlc-Glc). In the presence of Glc, dGlc6P reaches a plateau after 1 h or 2 h of incubation while the quantities of trehalose (Glc-Glc), dGlc-dGlc, dGlc-Glc, which are small at 1 h, rapidly increase with time. The reasons why dGlc and Glc exert opposite effects on their metabolism are discussed. The production of Glc-Glc decreases with increasing the external dGlc concentration or the dGlc/Glc ratio. The effect of dGlc on the biosynthesis of Glc-Glc can be explained by the competition of dGlc and Glc with respect to hexokinase. Although Glc favours the synthesis of dGlc6P, the maximum concentration of dGlc6P shows little dependence on the external dGlc concentration as long as glucose is not exhausted. The internal dGlc6P/dGlc ratio at equilibrium, about 4.7 +/- 0.7, is also found to be independent of the dGlc concentration in the suspension. Only a small fraction of dGlc6P disappears to give rise to the formation of dGlc-dGlc and dGlc-Glc. At equilibrium the inverse reaction from dGlc6P to dGlc may be important to compensate for the fast reaction of dGlc phosphorylation by hexokinase. At least nine series of experiments were conducted and showed that, in pyrophosphate media and for incubation times less than 4 h, dGlc-dGlc was not observed in the absence of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Non-cooperative effects of glucose and 2-deoxyglucose on their metabolism in Saccharomyces cerevisiae studied by 1H-NMR and 13C-NMR spectroscopy. 824 67

In this paper the substrate activities and binding affinities of the stereoisomers of the beta,gamma-bidentate Rh(H2O)4ATP and alpha,beta, gamma-tridentate Rh(H2O)3ATP complexes toward selected members of the kinase family of enzymes are reported. Hexokinase and glycerokinase were found to be specific for the delta beta, gamma-bidentate Rh(H2O)4ATP isomer as substrate while adenylate kinase was found to specifically catalyze the reaction of the delta beta,gamma-bidentate Rh(H2O)4ATP isomer. Pyruvate kinase recognized both the delta beta,gamma-bidentate Rh(H2O)4ATP isomer and the delta beta-P, exo alpha-P alpha,beta,gamma-tridentate Rh(H2O)3ATP isomer as substrates in the catalyzed phosphorylation of the alternate substrate, glycolate. 31P NMR analysis of the respective product complexes showed that alpha-P phosphoryl ligand exchange had not preceded or followed catalysis. Creatine kinase was found to be specific for the delta beta-P, exo alpha-P alpha,beta,gamma-tridentate Rh(H2O)3ATP isomer. Discrimination of the Rh(H2O)nATP isomers via preferential binding of the substrate-active isomer was observed for hexokinase and adenylate kinase but not for glycerokinase, fructose-6 phosphate kinase, creatine kinase, arginine kinase, or acetate kinase.
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PMID:Investigations of kinase substrate specificity with aqua Rh(III) complexes of adenosine 5'-triphosphate. 838 48

It has been proposed that in mammalian systems the glucose analog 2-fluoro-2-deoxy-D-glucose (FDG) is phosphorylated and subsequently converted to the corresponding mannose derivative via the action of phosphoglucose isomerase. As is generally true in metabolic studies of fluorinated molecules, the fluorine spectrum alone is suggestive, without providing definitive structural evidence, while the use of 1H NMR techniques generally suffers from a lack of adequate selectivity. A 1H-19F version of the hetero-RELAY experiment has been applied to this problem. Formation of the corresponding C-6 phosphorylated 2-FDG analog with hexokinase, followed by treatment of the resulting phosphorylated products with phosphoglucose isomerase, resulted in the observation of additional 19F resonances consistent with the corresponding 2-fluoro-2-deoxy-D-mannose-6-phosphate metabolite. A more definitive product identification was obtained using the hetero-RELAY experiment, which provides a complete 19F-decoupled proton spectrum for each of the fluorinated species.
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PMID:Identification of 2-fluoro-2-deoxy-D-glucose metabolites by 19F(1H) hetero-RELAY. 854 95

In this review, the results of a series of NMR experiments investigating glucose storage and synthesis in NIDDM patients and normal controls have been summarized. These have shown: 1. The deficit in nonoxidative glucose disposal in NIDDM subjects results from a defect in the muscle glycogen synthesis pathway. 2. Reduced activity of glucose transporter/hexokinase step in this pathway accounts for the reduced rate of glycogen synthesis in NIDDM patients. 3. This reduced activity of GT/Hk is a genetic defect present before the clinical onset of disease in prediabetic descendants of diabetic parents. 4. In muscle from normal, healthy subjects the rate of glycogen synthesis is controlled by the glucose transport/hexokinase activity step and not by the activity of the muscle glycogen synthase enzyme. 5. Hepatic gluconeogenesis is responsible for most hepatic glucose production during an overnight fast in both normal and NIDDM subjects, and increases in gluconeogenic flux are responsible for the increased rate of hepatic glucose production in NIDDM subjects. 6. In contrast to human muscle, where glycogenesis ceases at rest, in the liver gluconeogenesis and glycogenolysis are always active. Numerous previous studies were considered prior to embarking in each of these NMR experiments. In the original research articles we published, the earlier studies were discussed in terms of the relevant literature. Here, however, I have chosen to present the NMR data as simply as possible, in the hope of exposing the significance of these studies by disentangling the results from the complexities of NMR methodology.
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PMID:Nuclear magnetic resonance studies of glucose metabolism in non-insulin-dependent diabetes mellitus subjects. 889 70

31P NMR was used to measure the intracellular free magnesium concentration ([Mg2+]i) in human erythrocytes while [Mg2+]i was changed between 0.01 and 1.2 mM using the divalent cationophore A23187. 13C NMR and [2-13C]glucose were used to determine the kinetic effects of [Mg2+]i by measuring the flux through several parts of the glucose pathway. Glucose utilization was strongly dependent on [Mg2+]i, with half-maximal flux occurring at 0.03 mM. The rate-limiting step was most likely at phosphofructokinase, which has a Km(Mg2+) of 0.025 mM in the purified enzyme. Phosphorylated glycolytic intermediate concentration was also strongly dependent on [Mg2+]i and [MgATP], and glucose transport plus hexokinase may have been partially rate-determining at [Mg2+]i below approximately 0.1 mM. The pentose phosphate shunt activity was too low to determine the dependence on [Mg2+]i. Phosphoglycerate kinase and 2, 3-diphosphoglycerate mutase fluxes were also measured, but were not rate-limiting for glycolysis and showed no Mg2+ dependence. Human erythrocyte [Mg2+]i varies between 0.2 mM (oxygenated) and 0.6 mM (deoxygenated), well above the measured [Mg2+]i(1/2). It is unlikely, then, that [Mg2+]i plays a regulatory role in normal erythrocyte glycolysis.
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PMID:The regulatory role for magnesium in glycolytic flux of the human erythrocyte. 891 May 48

Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan. 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used to assess their rotational mobility in the intact cell. Comparison with the results obtained from relaxation time measurements of the purified enzymes in vitro and from theoretical calculations showed that two of the labeled enzymes, phosphoglycerate kinase and hexokinase, were tumbling in a cytoplasm that had a viscosity approximately twice that of water. There were no detectable signals from pyruvate kinase in vivo, although it could be detected in diluted cell extracts, indicating that there was some degree of motional restriction of the enzyme in the intact cell.
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PMID:19F NMR measurements of the rotational mobility of proteins in vivo. 899 36

The Embden-Meyerhof (EM) or Entner-Doudoroff (ED) pathways of sugar degradation were analyzed in representative species of the hyperthermophilic archaeal genera Thermococcus, Desulfurococcus, Thermoproteus, and Sulfolobus, and in the hyperthermophilic (eu)bacterial genus Thermotoga. The analyses included (1) determination of 13C-labeling patterns by 1H- and 13C-NMR spectroscopy of fermentation products derived from pyruvate after fermentation of specifically 13C-labeled glucose by cell suspensions, (2) identification of intermediates of sugar degradation after conversion of 14C-labeled glucose by cell extracts, and (3) measurements of enzyme activities in cell extracts. Thermococcus celer and Thermococcus litoralis fermented 13C-glucose to acetate and alanine via a modified EM pathway (100%). This modification involves ADP-dependent hexokinase, 6-phosphofructokinase, and glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAP:FdOR). Desulfurococcus amylolyticus fermented 13C-glucose to acetate via a modified EM pathway in which GAP:FdOR replaces GAP-DH/phosphoglycerate kinase. Thermoproteus tenax fermented 13C-glucose to low amounts of acetate and alanine via simultaneous operation of the EM pathway (85%) and the ED pathway (15%). Aerobic Sulfolobus acidocaldarius fermented 13C-labeled glucose to low amounts of acetate and alanine exclusively via the ED pathway. The anaerobic (eu)bacterium Thermotoga maritima fermented 13C-glucose to acetate and lactate via the EM pathway (85%) and the ED pathway (15%). Cell extracts contained glucose-6-phosphate dehydrogenase and 2-keto-3-deoxy-6-phosphogluconate aldolase, key enzymes of the conventional phosphorylated ED pathway, and, as reported previously, all enzymes of the conventional EM pathway. In conclusion, glucose was degraded by hyperthermophilic archaea to pyruvate either via modified EM pathways with different types of hexose kinases and GAP-oxidizing enzymes, by the nonphosphorylated ED pathway, or by a combination of both pathways. In contrast, glucose catabolism in the hyperthermophilic (eu)bacterium Thermotoga involves the conventional forms of the EM and ED pathways. The data are in accordance with various previous reports.
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PMID:Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium Thermotoga. 907 22

This work describes a search for hexokinase inhibitors based on the interactions analysis at the active site of the X-ray resolved o-tolulyl-glucosamine-hexokinase (OTG-HK) complex structure. As the actual enzyme sequence was unknown when the X-ray structure was made (only 30% homology), the structure of the complex was rebuilt by modelling on the X-ray structure frame which allowed residues in close vicinity to the inhibitor to be defined, particularly Glu249 and Gln278. Compounds with inhibitor-bearing groups able to interact with these residues were synthesized and assayed. Some of them revealed strong affinities, in the Km range for glucose. Kinetic analysis of their behaviour towards glucose and ATP together with spectroscopic studies using NMR, allowed the determination of the corresponding inhibition patterns and provided complementary information on HK.
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PMID:Yeast hexokinase inhibitors designed from the 3-D enzyme structure rebuilding. 924 53

The structure of adenine nucleotide bound at the active site of yeast hexokinase PII (PII) was studied in the complexes PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc using two-dimensional transferred NOE spectroscopy. Binding of the nucleotide ligand to the enzyme resulted in downfield shift of several ligand resonances. Changes in the chemical shift as a function of ligand concentration indicate that various resonances in the bound and free form of the ligand appear to be in fast exchange. The largest chemical shift change between the bound and the free states (delta vM = 88 +/- 9 Hz) at an NMR frequency of 500 MHz was observed for the H2 resonance of the adenine ring. A lower limit for the rate of ligand dissociation from the complex derived from these results is k(off) >> 550 s(-1). Interproton NOEs for various ligand protons in PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc complexes were measured at 500 MHz at 10 degrees C. The NOE buildup curves constructed from such measurements made at various mixing times (40, 80, 120, 160 and 200 ms) were analyzed using complete relaxation matrix calculations and various interproton distances were determined. These distances were used in restrained molecular dynamics calculations to derive the conformation of the nucleotide bound at the active site of the enzyme. The results of these calculations indicate that the nucleotide binds in an anti conformation. The glycosidic torsion angle chi (O4'-C1'-N9-C8) determined for the nucleotide in PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc complexes are 68 +/- 4 degrees, 52 +/- 4 degrees and 49 +/- 4 degrees respectively. In all these complexes, the ribose pucker is best represented by the unsymmetrical O4'-endo-C1'-exo twist ((o)T1). The observed decrease in the glycosidic torsion angle of the bound nucleotide is attributed to the glucose-induced conformational changes in the enzyme. The structure of the nucleotide derived here is at variance from the one proposed on the basis of X-ray crystallography and model building [Shoham, M. & Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14].
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PMID:Yeast hexokinase PII--bound nucleotide conformation at the active site. 942 8

The conformation of ADPMg(II) bound at the active site of yeast hexokinase PI has been determined using transferred nuclear Overhauser effect spectroscopy (TRNOESY). We have measured the time dependent NOE buildup of all the proton pairs of ADP in enzyme. ADPMg(II) and enzyme. glucose.ADPMg(II) complexes at 500 MHz and 10 degrees C. The data have been analyzed using complete relaxation matrix approach to obtain various inter-proton distances. These distances were used as restraints in the molecular dynamics and energy minimization to obtain the conformation of the bound nucleotide. The results from these calculations suggest that in both the complexes, the nucleotide binds in an anti conformation with a glycosidic torsion angle chi = 55 +/- 5 degrees and 52 +/- 5 degrees in PI.ADPMg(II) and PI.glucose.ADPMg(II) complexes, respectively. However, the phase angle of pseudorotation (P) which defines the sugar pucker in the two complexes was found to be 99 degrees, corresponding to 0T1 for PI.ADPMg(II) complex and 69 degrees corresponding to 4T0 for PI.glucose.ADPMg(II) complex. The cleft closure conformational change in the enzyme induced by glucose seems to affect the conformation of the ribosyl moiety of the bound nucleotide.
Physiol Chem Phys Med NMR 1998
PMID:Effect of glucose on the conformation of ADPMg(II) bound at the active site of yeast hexokinase PI. 980 35


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