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)

Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6-[N-(6-aminohexyl)carbamoyl]-ADP, N6-[N-(6-aminohexyl)carbamoyl]-ATP, and N6-[N-(6-aminohexyl)carbamoyl]-AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP. Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr-activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6-phosphate. A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr-activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.
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PMID:Preparation of N6-[N-(6-aminohexyl) carbamoyl]-adenine nucleotides and their application to coenzymically active immobilized ADP and ATP, and affinity adsorbents. 19 56

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.
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PMID:Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase. 114 Jan 97

The functionally diverse actin, hexokinase, and hsp70 protein families have in common an ATPase domain of known three-dimensional structure. Optimal superposition of the three structures and alignment of many sequences in each of the three families has revealed a set of common conserved residues, distributed in five sequence motifs, which are involved in ATP binding and in a putative interdomain hinge. From the multiple sequence alignment in these motifs a pattern of amino acid properties required at each position is defined. The discriminatory power of the pattern is in part due to the use of several known three-dimensional structures and many sequences and in part to the "property" method of generalizing from observed amino acid frequencies to amino acid fitness at each sequence position. A sequence data base search with the pattern significantly matches sugar kinases, such as fuco-, glucono-, xylulo-, ribulo-, and glycerokinase, as well as the prokaryotic cell cycle proteins MreB, FtsA, and StbA. These are predicted to have subdomains with the same tertiary structure as the ATPase subdomains Ia and IIa of hexokinase, actin, and Hsc70, a very similar ATP binding pocket, and the capacity for interdomain hinge motion accompanying functional state changes. A common evolutionary origin for all of the proteins in this class is proposed.
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PMID:An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. 132 28

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.
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PMID:Radiometric assays for glycerol, glucose, and glycogen. 281 33

A method has been developed for calculating rate constants for dehydration of aldehydes that induce ATPase reactions by kinases and where 18O is transferred from the aldehyde or its hydrate to inorganic phosphate during the reaction. The method involves measurement of the fraction of 18O in phosphate by 31P NMR after the ATPase reaction has proceeded for several minutes with zero-order kinetics. The reaction is started by addition of the aldehyde in a small volume of H2 18O, and the speed of washout of 18O by reversible dehydration relative to the rate of the ATPase reaction allows calculation of the rate constants if the hydration equilibrium constant is known from the proton NMR spectrum of the aldehyde. Dehydration rate constants (s-1 at pH 8-8.5, 0.1 M buffer, 25 degrees C) for the following aldehydes (all over 95% hydrated) and kinases used are as follows: D-glyceraldehyde with glycerokinase, 0.03; 2,5-anhydro-D-mannose 6-phosphate with fructose-6-phosphate kinase, 0.025; 2,5-anhydro-D-mannose or 2,5-anhydro-D-talose with fructokinase, 0.029 and 0.017, respectively; D-gluco-hexodialdose with hexokinase, 0.068. With betaine aldehyde and choline kinase or glyoxylate and pyruvate kinase, no 18O was transferred to phosphate during the ATPase reactions. However, the dehydration rate constant for glyoxylate (0.007 s-1 at pH 7 extrapolated to zero buffer concentration and up to 0.11 s-1 at pH 9.0 with 0.3 M buffer) was determined by extrapolating the initial rate of reduction of the free aldehyde catalyzed by lactate dehydrogenase to infinite enzyme levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel method for determining rate constants for dehydration of aldehyde hydrates. 609 90

Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the presence of keto or aldehyde analogues (N-methylhydantoic acid, D-glyceraldehyde 3-phosphate, and acetaldehyde, respectively), possibly because of the absence of an acid-base catalytic group in the latter two cases. These analogues were competitive inhibitors vs. the normal substrates, and in the latter case, the hydrate of acetaldehyde was shown to be the inhibitory species on the basis of the deuterium isotope effect on the inhibition constant.
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PMID:Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases. 609 91

By introducing fructose into the glycolysis, it is possible to stimulate ATP formation. As is the case in animal experiments, in human lenses, too, the first step in the phosphorylation to fructose-1-phosphate via the enzyme ketohexokinase. The present investigation deals with the question whether enzymes present in the lens are responsible for the further steps in fructose degradation. Particularly the aldolase isoenzyme C splits fructose-1-phosphate into glyceraldehyde and dihydroxyacetone phosphate in the same way as in glucose catabolism. Dihydroxyacetone phosphate can further be directly degraded and thus utilized to ATP formation. From glyceraldehyde, glycerol (aldose reductase) or glycerate (aldehyde dehydrogenase) can be formed. The presence of triosekinase, which phosphorylates glyceraldehyde directly to glyceraldehyde-3-phosphate, could only be determined in the lens tissue of young animals. The presence of glycerokinase (glycerol leads to glycerophosphate) could not be verified. Thus, in the lens tissue 1 ATP molecule net per fructose molecule can be formed. In older age, the glucose breakdown is limited by hexokinase and phosphofructokinase, so that the glucose, after transformation via the sorbitol pathway to fructose, can also be utilized for the energy metabolism.
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PMID:Investigations of the enzymes involved in the fructose breakdown in the cattle lens. 628 47

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

We have developed an uncoupled, pH sensitive kinase assay that can be used for high-throughput screening of potential inhibitors or for determining substrate specificity. Kinases catalyze the transfer of a gamma-phosphoryl group from ATP to an appropriate hydroxyl acceptor with the release of a proton. This assay is based on the detection of this proton using an appropriately matched buffer/indicator system. The assay was used to measure the activity of four readily available kinases, including hexokinase, glucokinase, glycerokinase, and pyruvate kinase, which was run in the reverse direction. We also went on to screen a small series of mono- and diphosphonucleotides for inhibition of hexokinase as well as a modest set of potential hexokinase substrates. We determined sucrose to be a modest substrate for hexokinase with a K(m) of 1.8 +/- 0.2 mM, a k(cat) of 142 +/- 3 min(-1), and a V(max) that is 15% of that for glucose. Given the importance of kinases in a diverse array of biological functions and disease states, there is a need for a simple, rapid assay system. We feel this assay will lend itself well to meet this end. This method should be applicable to many other enzymatic reactions which involve a change in pH.
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PMID:A pH sensitive colorometric assay for the high-throughput screening of enzyme inhibitors and substrates: a case study using kinases. 1181 41

The active and selective transport of glucose and glycerol was carried out using electrophoresis and artificial enzymatic membranes. These positively charged composite membranes carry, on the face adjacent to the donor compartment of an electrophoresis module, a specific kinase (hexokinase or glycerokinase) and, on the opposite face, an alkaline phosphatase (ALP). Phosphorylation of the neutral substrate (glucose or glycerol) on the donor side by the kinase generates a negatively charged phosphorylated substrate, whose transmembrane migration is promoted by an electric field and by the membrane's positive charge. Dephosphorylation of the phosphorylated substrate by ALP on the opposite face regenerates the neutral substrate, which accumulates in the receiver compartment of the electrophoresis module. Using an electrophoresis module specifically designed for this study, our experiments were carried out enabling glucose and glycerol to be concentrated approximately eight- and twelve-fold, respectively, in 8 h.
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PMID:Enzymatic membranes for the selective transport of neutral molecules by electrophoresis. 1843


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