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)

Hexokinase from pyloric caeca of the starfish, Asterias amurensis, was purified to a specific activity of 148 units/mg protein. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular weight determined by SDS polyacrylamide gel electrophoresis and Ultrogel AcA 34 gel filtration was about 50,000. The enzyme showed a broad pH optimum ranging from 7.4 to 9.5. The Km values for D-glucose, D-fructose, 2-deoxy-D-glucose, D-mannose, D-glucosamine and ATP were 0.045, 4, 0.21, 0.05, 0.35 and 0.3 mM, respectively. N-Acetyl-D-glucosamine, D-xylose and D-galactose were not phosphorylated. The enzyme was strongly inhibited by the reaction products, glucose 6-phosphate and ADP, but not by high levels of D-glucose. The starfish hexokinase thus resembled mammalian isozyme A with respect to kinetic properties.
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PMID:Purification and properties of hexokinase from the starfish, Asterias amurensis. 89 76

By using competition between glucose and its analogue D-glucosamine, we have produced a system in which it is possible to vary the steady-state growth rate of populations of Saccharomyces cerevisiae cells without otherwise altering the composition of the medium or significantly affecting catabolite repression. We demonstrate that D-glucosamine inhibits the accumulation of glucose derived label and the phosphorylation of glucose by hexokinase (EC 2.7.1.1).
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PMID:Controlling the growth rate of Saccharomyces cerevisiae cells using the glucose analogue D-glucosamine. 269 46

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 glucose analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a "specific" pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.
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PMID:Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme. 357 10

A reactive Glc analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), has recently been used (D. M. Schirch and J. E. Wilson (1987) Arch. Biochem. Biophys. 254, 385-396) to label the Glc binding site of rat brain Type I hexokinase. This site has been located in a 40-kDa domain at the C-terminus of the enzyme previously shown to be the location of the substrate ATP binding site (M. Nemat-Gorgani and J. E. Wilson (1986) Arch. Biochem. Biophys. 251, 97-103). In the present study, peptide mapping of hexokinase modified by radiolabeled GlcNBrAc yields three labeled peptides (Peptides I-III). Peptides I and III, as well as catalytic activity, are protected by inclusion of Glc or GlcNAc during reaction with GlcNBrAc. These two peptides show considerable homology to contiguous regions in the sequences of yeast hexokinase isozymes A and B. Peptide III is homologous to a sequence which, based on the X-ray crystallographic work by Steitz and co-workers, is located near the Glc binding site of yeast hexokinase; Peptide I is homologous to an immediately adjacent (toward the C-terminus) region of yeast hexokinase. An essential serine residue implicated in the binding of Glc to the yeast enzyme is also conserved in Peptide III from rat brain hexokinase. These results provide strong support for the view that the "catalytic domain" at the C-terminus of the mammalian Type I hexokinase shares a common ancestry with yeast hexokinase. Peptide II appears to be nonspecifically labeled by GlcNBrAc since labeling is insensitive to the presence of protective ligands such as Glc or GlcNAc; the sequence of Peptide II shows no detectable homology with the yeast isozymes.
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PMID:Rat brain hexokinase: amino acid sequence at the substrate hexose binding site is homologous to that of yeast hexokinase. 363 58

We have compared the competitive inhibitory effects of 2-deoxyglucose, glucosamine, N-acetylglucosamine, N-benzoylglucosamine, and the commonly used radiographic and density gradient agent metrizamide (2-[3-acetamido-2,4,6-triiodo-5-(N-methylacetamido) benzamido]-2-deoxyglucose) on the mitochondrial and soluble forms of human brain hexokinase. Metrizamide produces a classical competitive inhibition with glucose for human brain hexokinase, with Kis of 2.8 and 2.5 mM, respectively, for the mitochondrial and soluble forms. Glucosamine exhibited Kis of 0.58 and 0.29 mM, while 2-deoxyglucose exhibited Kis of 0.074 and 0.15 mM and N-acetylglucosamine 0.098 and 0.092 mM for these two forms, respectively. N-Benzoylglucosamine was by far the most effective inhibitor tested, with Ki values of 0.0086 and 0.022 mM, respectively. In order of increasing potency as a competitive inhibitor for mitochondrial hexokinase are metrizamide, glucosamine, N-acetylglucosamine, 2-deoxyglucose, and N-benzoylglucosamine. For the soluble form of the enzyme in increasing potency are metrizamide, glucosamine, 2-deoxyglucose, N-acetylglucosamine, and N-benzoylglucosamine. Since N-benzoylglucosamine was over 100 times more potent than metrizamide, some of the effects of metrizamide could be due to contamination by N-benzoylglucosamine. However, gas chromatography-mass spectrometry analysis of metrizamide did not indicate the presence of N-benzoyl-glucosamine. In addition, column chromatographic separation of commercially available metrizamide and reconstitution of freeze-dried eluate fractions localized the inhibitory effect to the metrizamide peak.
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PMID:Competitive inhibition of human brain hexokinase by metrizamide and related compounds. 669 84

An analogue of the substrate glucose, N-(bromoacetyl)-D-glucosamine (GlcNBrAc) inactivates bovine brain mitochondrial hexokinase completely and irreversibly in a pseudo-first-order fashion at pH 8.5 and 22 degrees C. The rate of inactivation of hexokinase by this reagent does not increase linearly with increasing reagent concentration but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. The pH dependence of the rate of inactivation suggests that a group on the enzyme with pKa = 9.1 is being modified by this reagent. At pH 8.0 the rate of inactivation by this reagent is very slow, and it can be shown to be a competitive inhibitor of the hexokinase reaction with respect to the substrate glucose. The substrates glucose and ATP strongly protected the enzyme against the inactivation reaction. The inactivation of the enzyme was found to be accompanied by the alkylation of two sulfhydryl residues as shown by the formation of approximately 2 mol of S-(carboxymethyl)-cysteine/mol of inactivated enzyme. Treatment of the enzyme with 14C-labeled reagent results in the incorporation of approximately 2 mol of reagent/mol of inactivated enzyme. However, the enzyme protected by glucose still shows the incorporation of approximately 1 mol of the labeled reagent/mol of the enzyme. From a tryptic digest of the enzyme inactivated by this reagent, two labeled peptides were obtained, one of which was absent if the labeling reaction was carried out in presence of glucose. These results indicate that the affinity reagent reacts with two thiols, only one of which is crucial for the activity of the enzyme and is located in the region of its active site.
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PMID:Reaction of brain hexokinase with a substrate-like reagent. Alkylation of a single thiol at the active site. 740 81

Human beta-cell glucokinase recognition and phosphorylation of different sugars was investigated by steady-state kinetic analysis, measurements of substrate-induced intrinsic fluorescence changes, and molecular modeling and calculation of interaction energies. Measurements of kcat/Km showed that glucokinase phosphorylated the sugars in the order glucose = mannose > deoxyglucose > fructose = glucosamine. The mode of binding of these sugars to the open conformation of glucokinase was predicted from molecular modeling. Glucokinase is predicted to form similar interactions with the 6-OH, 4-OH, and 1-OH groups of all these sugars. The interactions of the 2-OH and 3-OH groups differ and depend on the type of sugar and reflect differences in cooperative behavior. For example, glucose and deoxyglucose exhibited cooperative behavior with Hill coefficients of 1.8 and 1.5, respectively, while mannose and fructose demonstrated Michaelis-Menten behavior. Galactose, allose, and 2,5-anhydroglucitol were not substrates under the assay conditions used, and the alpha- and beta-anomers of methylglucose were poor substrates with Km's greater than 1000 mM. Glucokinase exhibited an ATPase activity which was 1/2000th that of the rate of the kinase reaction, and unlike yeast hexokinase, it was not affected by the addition of lyxose. Glucosamine was a low affinity inhibitor as well as a substrate, while N-acetylglucosamine and mannoheptulose were high-affinity inhibitors. The change in intrinsic fluorescence that was induced by glucose, mannose, and mannoheptulose had the opposite sign for glucosamine, which implies a very different mode of binding from the other sugars. The calculated interaction energies of glucokinase with glucose, mannose, deoxyglucose, and fructose agree very well with the measured values of kcat/Km, which indicates that these sugars are recognized by binding to the open conformation of glucokinase.
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PMID:Sugar specificity of human beta-cell glucokinase: correlation of molecular models with kinetic measurements. 774 12

Glucosamine, a potent inhibitor of glucokinase (hexokinase IV or D), was used to estimate the contribution of this enzyme to glucose phosphorylation in freshly isolated rat hepatocytes and its sensitivity to fructose 6-phosphate in situ. Experiments with radiolabelled glucosamine indicated that this amino sugar, at concentrations of 5 or 40 mM, readily penetrated hepatocytes to reach in 1 min a total (i.e., glucosamine+metabolites) intracellular concentration equal to 0.8-1.2-fold its extracellular concentration. In marked contrast, N-acetylglucosamine barely penetrated the cells. The detritiation of [2-3H]glucose, used to estimate glucose phosphorylation in intact cells, was inhibited by glucosamine much more potently than by N-acetylglucosamine, half-maximal effects being reached at about 2.5 and 30 mM respectively. Extrapolation of the data indicated that about 12% of the detritiation was resistant to glucosamine. Dihydroxyacetone (10 mM), lactate (10 mM) + pyruvate (1 mM), and glucagon (1 microM) increased up to 8-fold the concentration of hexose 6-phosphates (glucose 6-phosphate+fructose 6-phosphate) and, against expectations, modestly decreased the detritiation rate measured in the absence of glucosamine. In the presence of 40 mM glucosamine, these agents increased the detritiation rate, which then positively correlated with the concentration of hexose 6-phosphates. This hexose 6-phosphates-dependent detritiation was sensitive to inhibition by vanadate, and was also catalysed by gel-filtered cell-free extracts, as well as by liver microsomes in the presence of phosphoglucoisomerase; it can be explained by an exchange reaction catalysed by glucose-6-phosphatase. When this exchange reaction is taken into account, it appears that the rate of glucose detritiation attributable to glucokinase decreases when the concentration of hexose 6-phosphates increases. This is in agreement with the known effect of fructose 6-phosphate to potentiate the inhibition of glucokinase by its regulatory protein.
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PMID:Glucosamine-sensitive and -insensitive detritiation of [2-3H]glucose in isolated rat hepatocytes: a study of the contributions of glucokinase and glucose-6-phosphatase. 775 69

N-Acetyl-D-[2-3H]glucosamine was synthesized from N-acetyl-D-mannosamine by alkaline 2-epimerization in pyridine containing 3H2O and nickelous acetate. The reaction involves reversible formation of an enol intermediate and therefore also resulted in incorporation of tritium into N-acetylmannosamine. After completed reaction, the two N-acetylhexosamines were separated from other radioactive products and Morgan-Elson chromogens by chromatography on a column of Sephadex G-10, which was eluted with 10% ethanol, and were then separated from each other by chromatography on Sephadex G-15 in 0.27 M sodium borate (pH 7.8). The location of the incorporated tritium was established by treatment of the N-acetylhexosamines with borate under the conditions of the Morgan-Elson reaction, which converts the sugars to Kuhn's chromogen I with concomitant loss of the C-2 hydrogen. As expected, this treatment resulted in the formation of 3H2O, indicating that the tritium was located at C-2. [2-3H]Glucosamine was prepared by acid hydrolysis of the labelled N-acetylglucosamine and was converted to [2-3H]glucosamine 6-phosphate by incubation with hexokinase and ATP. The sugar phosphate was used as a substrate for glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10) in a simple 3H2O release assay.
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PMID:Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water. 778 Jan 91


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