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 specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.
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PMID:The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei. 292 7

Past work, including our computer simulation of cardiac energy metabolism, indicates that magnesium is an important coherent controller of glycolysis and the Krebs cycle. Many of the glycolytic enzymes are sensitive to Mg2+. The most important effect is due to MgATP2-being a cofactor for a number of these enzymes while other chelation forms are inactive or inhibitory. The means by which Mg2+ and Mg2+ chelates of adenine nucleotides regulate the most important glycolytic enzymes--hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, and pyruvate kinase--are described in detail. Creatine kinase, which is important in energy metabolism and highly sensitive to both metal ions and pH, is also discussed. It is necessary to properly control the composition of assay mixtures (particularly with regard to metal ions) in order to determine what actually regulates the activity of an enzyme.
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PMID:Magnesium regulation of the glycolytic pathway and the enzymes involved. 293 60

A new adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), has been synthesized. The effectiveness of PLP-AMP as an affinity probe has been tested using a number of nucleotide-binding enzymes. In comparison to reaction with pyridoxal 5'-phosphate, PLP-AMP binds more tightly and exhibits greater specificity of labeling for most enzymes tested. PLP-AMP is a very potent inhibitor of yeast alcohol dehydrogenase and rabbit muscle pyruvate kinase, with complete inhibition obtained upon incorporation of 1 mol of reagent/mol of catalytic subunit. The reagent is also a potent inhibitor of yeast hexokinase and phosphoglycerate kinase. When modified in the absence of substrates, these enzymes require 2 mol of reagent/mol of active site for complete inhibition. However, when modified in the presence of sugar substrates, this stoichiometry decreases to 1.1 for the hexokinase-glucose complex and 1.4 for the phosphoglycerate kinase . 3-phosphoglycerate complex. The most potent inhibition by PLP-AMP was observed with rabbit muscle adenylate kinase. Half-maximal inhibition was obtained at a concentration of approximately 1 microM. In contrast to these examples, PLP-AMP, as well as pyridoxal 5'-phosphate, fails to act as a potent or specific inhibitor of beef heart mitochondrial F1-ATP-ase. The high specificity of labeling and the ability of nucleotide substrates to decrease the rate of inactivation of the kinases and dehydrogenase are consistent with the modification of active site residues. The complete reversibility of both modification and inactivation in the absence of reduction by NaBH4 and the absorption spectra of modified enzymes prior to and following reduction indicate reaction with lysyl residues. We conclude that PLP-AMP holds considerable promise as an affinity label for exploring the structure and mechanism of nucleotide-binding enzymes.
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PMID:Affinity labeling of nucleotide-binding sites on kinases and dehydrogenases by pyridoxal 5'-diphospho-5'-adenosine. 293 39

We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8-55%. Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM- and DEAE-cellulose) which resulted in the purification of all nine enzymes. The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80-90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1-5 kDa larger in size. Together these nine enzymes contribute 3.3 +/- 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants. A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with pI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3-6 higher than in the case of the various unicellular organisms. It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).
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PMID:Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties. 294 90

Enthalpies of binding of MgADP, MgATP, and 3-phosphoglycerate to yeast phosphoglycerate kinase have been determined by flow calorimetry at 9.95-32.00 degrees C. Combination of these data with published dissociation constants [Scopes, R.K. (1978) Eur. J. Biochem. 91, 119-129] yielded the following thermodynamic parameters for the binding of 3-phosphoglycerate at 25 degrees C: delta Go = -6.76 +/- 0.11 kcal mol-1, delta H = 3.74 +/- 0.08 kcal mol-1, delta So = 35.2 +/- 0.6 cal K-1 mol-1, and delta Cp = 0.12 +/- 0.32 kcal K-1 mol-1. The thermal unfolding of phosphoglycerate kinase in the absence and presence of the ligands listed above was studied by differential scanning calorimetry. The temperature of half-completion, t 1/2, of the denaturation and the denaturational enthalpy are increased by the binding of the ligands, the increase in t 1/2 being a manifestation of Le Chatelier's principle and that in enthalpy reflecting the enthalpy of dissociation of the ligand. Only one denaturational peak was observed under all conditions, and in contrast with the case of yeast hexokinase [Takahashi, K., Casey, J.L., & Sturtevant, J.M. (1981) Biochemistry 20, 4693-4697], no definitive evidence for the unfolding of more than one domain was obtained.
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PMID:Thermodynamic study of yeast phosphoglycerate kinase. 354 15

In the process of defining the recruitment of fuel and pathway selection in rainbow trout fast-twitch white skeletal muscle, it was clear that the near-maximal myosin adenosinetriphosphatase activity during a 10-s sprint was supported solely by phosphocreatine hydrolysis. A conservative estimate of the ATP turnover was 188 mumol X g wet wt-1 X min-1. It was not until the rate and force of contraction decreased that the relative contribution of anaerobic glycogenolysis became increasingly important. Over a 10-min period of burst swimming at approximately 120% of maximum aerobic steady-state swimming velocity of trout determined in a Brett-type swim tunnel, fatigue was associated with the near-depletion of glycogen in white muscle. The ATP turnover supported by anaerobic glycogenolysis was 78 mumol X g wet wt-1 X min-1. The glycolytic pathway appeared functional at this time with control sites being identified at hexokinase and phosphofructokinase (PFK-1). PFK-1 did not appear to be inhibited by low muscle pH (pH 6.66). In another exercise protocol lasting 30 min, complete exhaustion was related to glycogen depletion. The sum of all glycolytic intermediates from glucose 6-phosphate to pyruvate at exhaustion decreased by a dramatic 80% compared with the 25% decrease for the 10-min fatigue swimming protocol. This large depletion of glycolytic intermediates was accompanied by an 80% fall in ATP, a 70-80% reduction in the ATP/ADP and phosphorylation potential, and a 2.5-fold increase in the NAD/NADH. Associated with these changes was a marked displacement of the phosphoglycerate kinase (PGK), and the combined glyceraldehyde-3-phosphate dehydrogenase-PGK reactions from thermodynamic equilibrium. As a general conclusion, fatigue and exhaustion should be viewed as a multicomponent biochemical process in response to low glycogen and not leveled at one particular step of the glycolytic pathway.
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PMID:Regulation of anaerobic ATP-generating pathways in trout fast-twitch skeletal muscle. 360 83

The glucose consumption rate versus ATP content in human red cells (regulatory patterns of glycolysis) and ATP concentration versus glucose uptake rate in red cell suspension (regulatory patterns of total ATPases), when the rate of glucose uptake is constant and lower than the rate of glucose consumption at physiological conditions, were measured at different pH values. The shape of both types of kinetic curves was found to be dependent on the pH of the incubation medium but the same for the red cells taken from different donors. It is supposed that at alkaline pH, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions become the rate-limiting steps of glycolysis instead of hexokinase and phosphofructokinase under physiological conditions.
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PMID:[Effect of pH on the regulatory characteristics of energy metabolism in human erythrocytes]. 376 40

The glycosomes of in vitro grown procyclic trypomastigote forms of Trypanosoma brucei were purified by three different procedures and the results compared by electron microscopy, enzyme assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Centrifugation on a self-forming Percoll gradient followed by a sucrose gradient centrifugation resulted in the least enriched glycosomal preparation. Centrifugation on a pre-formed Nycodenz gradient gave an improved preparation but the most homogeneous preparation of intact glycosomes was obtained after centrifugation on two successive sucrose gradients. Glycosomes purified by both the Nycodenz and double sucrose gradient procedures appeared larger than in situ glycosomes presumably due to an osmotic effect resulting from disruption of the granular matrix of the organelles. Nevertheless, there appears to be no loss of cisternal contents due to the swelling of the organelles. The glycosomes of the bloodstream form trypomastigotes purified by the same procedures show, however, no sign of swelling. A comparison of glycosomes purified from procyclic trypomastigotes and bloodstream form trypomastigotes prepared by the same double sucrose procedure demonstrated that in the glycosome of procyclic trypomastigotes: activities of hexokinase, phosphoglucose isomerase, phosphofructose kinase, aldolase and phosphoglycerate kinase and diminished by 80-100%; activities of glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and glycerol-3-phosphate dehydrogenase remain unchanged or are only slightly reduced; there is an appearance of four major new proteins, among which could be phosphoenol pyruvate carboxykinase and malate dehydrogenase. These observations are in basic agreement with those by Hart et al. (Mol. Biochem. Parasitol. 12, 25-35, 1984).
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PMID:An improved purification of glycosomes from the procyclic trypomastigotes of Trypanosoma brucei. 380 43

The enzyme activities of cultured early erythroid progenitor cells (burst-forming unit erythroid, BFU-E) were measured and were compared with the activities of mature erythrocytes. The enzyme activity of acetylcholinesterase was not detectable in the erythroblasts. The ratios of phosphofructokinase and glutathione peroxidase were low due to low enzyme activities in both the erythroblasts and erythrocytes. The ratios of triose phosphate isomerase, phosphoglycerate kinase, and adenylate kinase were low due to high enzyme activities in both the erythroblasts and erythrocytes. The ratios of hexokinase, glucose phosphate isomerase, monophosphoglyceromutase, pyruvate kinase, and adenosine deaminase were high due to high enzyme activities in the erythroblasts. The isozyme of erythroblast hexokinase was of the prototype isozyme I, while pyruvate kinase was predominantly of the prototype M2, with two hybrid isozymes to the anodal side by electrophoresis. These facts suggest that there is a greatly different metabolic pattern during the maturation of the erythroid cells.
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PMID:Enzyme activities of cultured erythroblasts. 403 55

The storage lesion which limits the shelf life of human blood in blood banking is associated with a metabolic loss of 2,3-diphosphoglycerate and ATP. This metabolic loss is driven by intracellular ATPase which are usually considered to include the ion pumps and the reactions which maintain the discoid shape of the human erythrocyte. Under the acidic conditions of blood storage, the energy-yielding reactions of the glycolytic pathway are restricted at the hexokinase and phosphofructokinase steps. We show here that under such circumstances the enzyme of the diphosphoglycerate shunt, diphosphoglycerate mutase/phosphatase and the glycolytic enzyme phosphoglycerate kinase can form a futile cycle with ATPase activity. This ATPase activity responds to 2-phosphoglycolate which is known to activate both diphosphoglycerate mutase and diphosphoglycerate phosphatase reactions. When the enzymes of the futile cycle are combined with the enzymes of the lower glycolytic pathway in a reconstitution experiment designed to represent conditions within the stored erythrocyte, the futile cycle does provide an ATPase activity which results in the metabolic loss of 2,3-diphosphoglycerate. An isotope incorporation experiment demonstrates that the futile cycle is active in glucose-depleted erythrocytes.
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PMID:A futile cycle in erythrocyte glycolysis. 406 53


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