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)

DNA polymorphisms in the glucokinase gene have recently been shown to be tightly linked to early-onset non-insulin-dependent diabetes mellitus in approximately 80% of French families with this form of diabetes. We previously identified a nonsense mutation in exon 7 in one of these families and showed that it was the likely cause of glucose intolerance in this dominantly inherited disorder. Here we report the isolation and partial sequence of the human glucokinase gene and the identification of two missense mutations in exon 7, Thr-228----Met and Gly-261----Arg, that cosegregate with early-onset non-insulin-dependent diabetes mellitus. To assess the molecular mechanism by which mutations at these two sites may affect glucokinase activity, the crystal structure of the related yeast hexokinase B was used as a simple model for human beta-cell glucokinase. Computer-assisted modeling suggests that mutation of Thr-228 affects affinity for ATP and mutation of Gly-261 may alter glucose binding. The identification of mutations in glucokinase, a protein that plays an important role in hepatic and beta-cell glucose metabolism, indicates that early-onset non-insulin-dependent diabetes mellitus may be primarily a disorder of carbohydrate metabolism.
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PMID:Human glucokinase gene: isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus. 150 86

We studied the possible relationships between the functional status of the beta-cell and activities or mRNA contents of enzymes involved in the catabolism of glucose. Three different in vitro models with attenuated insulin response were used: rat islets cultured at a low glucose concentration, rat islets incubated in vitro with streptozocin, and fetal rat islets. The fetal and streptozocin-administered islets were compared with adult islets cultured in RPMI-1640 containing 11 mM glucose, and the effects of the in vitro glucose concentrations (3.3, 11, and 28 mM) were assessed on adult islets only. Cellular mRNA levels for the mitochondrial DNA-encoded cytochrome b and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by Northern-blot analysis. Enzymatic activities of high-Km (glucokinase) and low-Km (hexokinase) glucose-phosphorylating enzymes and succinate-cytochrome c reductase were also determined. Islets cultured at 3.3 mM glucose displayed a decreased activity of glucokinase compared with islets cultured at 28 mM glucose (23.3 +/- 12%), whereas there was no difference in hexokinase activity or the level of GAPDH mRNA. The activity of succinate-cytochrome c reductase was similar in islets cultured at the different glucose concentrations. The level of cytochrome b mRNA increased at 28 mM glucose compared with islets cultured at 11 mM glucose (140 +/- 14%). Islets incubated with streptozocin and subsequently cultured for 7 days at 11 mM glucose exhibited a decreased level of cytochrome b mRNA (65 +/- 5%) and no differences in the activities of glucokinase, hexokinase, succinate-cytochrome c reductase, or the level of GAPDH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Exhibition of specific alterations in activities and mRNA levels of rat islet glycolytic and mitochondrial enzymes in three different in vitro model systems for attenuated insulin release. 164 83

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) was covalently coupled to collagen-coated silica gel beads. The immobilized enzyme, as a packed-bed reactor, was used to determine glucose in serum and other samples. The method was based on a spectrophotometric measurement of NADPH produced by two consecutive reactions, similar to the hexokinase method. The described approach takes advantage of the greater stability of polyphosphate compared to that of ATP, the greater specificity of polyphosphate glucokinase versus that of hexokinase, and the reusability of the immobilized enzyme. Linearity, precision, and accuracy of the method were tested and found to be very good. The results were linear between 10 and 50 nmol of glucose in a 50-microliter sample and the coefficient of variation was less than 4% in five successive determinations. The recovery of glucose was about 100% after calibration of the method. The results of the measurements correlated well with those obtained with soluble polyphosphate glucokinase (r = 0.997, y = 1.036x - 0.016). The immobilized-enzyme reactor showed good operational stability during a month of use, losing about 12% of its initial activity.
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PMID:Glucose determination using immobilized polyphosphate glucokinase. 166 65

We have investigated the mechanism by which the replacement of a Na(+)-rich medium by a K(+)-rich medium causes an increase in the apparent affinity of glucokinase (hexokinase IV or D) for glucose in isolated hepatocytes [Bontemps, F., Hue, L. & Hers, H. G. (1978) Biochem. J. 174, 603-611]. The stimulatory effect of a K(+)-rich medium on the rate of glucose phosphorylation, as assessed by the release of tritium from [2-3H]glucose, was only partially additive with the effect of fructose, suggesting that it was also due to a decrease in the inhibition exerted on glucokinase by its regulatory protein. Measurements of metabolites indicated that the effect of the K(+)-rich medium was neither due to the formation of fructose 1-phosphate, nor to changes in the concentrations of fructose 6-phosphate or Pi, two other effectors of the regulatory protein. Replacement of Na+ by K+ in the medium resulted in a time-dependent and dose-dependent increase in cell volume that paralleled the changes in the rate of detritiation observed at 5 mM glucose. The water and chloride contents, estimated using radiolabelled compounds, were threefold and tenfold higher, respectively, in K+ cells than in Na+ cells, and the intracellular Cl- concentration about threefold higher (94 versus 29 meq/l). The effects of the K(+)-rich medium on cell volume, Cl- concentration and rate of detritiation were greatly reduced by including 80 mM trehalose or sucrose in the medium at the start of the incubation. Addition of trehalose to cells incubated for 45-50 min in the K(+)-rich medium caused an immediate decrease in cell volume whereas the rate of detritiation and the Cl- concentration underwent a transient increase followed by a decrease. Replacement of KCl by KBr, potassium acetate or potassium trichloroacetate in the K(+)-rich medium resulted in different relationships between cell volume and the rate of detritiation, in agreement with the differential effect of these salts on the activity of purified glucokinase assayed in the presence of regulatory protein. From these results we conclude that the increase in the activity of glucokinase induced by a KCl-rich medium is at least partly due to an increase in the concentration of Cl-, which relieves the inhibition exerted by the regulatory protein on purified glucokinase.
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PMID:Mechanism of the stimulatory effect of a potassium-rich medium on the phosphorylation of glucose in isolated rat hepatocytes. 174 Jan 48

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.
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PMID:Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin). 183 54

Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.
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PMID:Expression and site-directed mutagenesis of hepatic glucokinase. 185 32

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.
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PMID:Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII. 186 42

A human liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) cDNA was isolated from a liver cDNA library. This cDNA (hLGLK1) appeared to be full length [2548 base pairs (bp) plus additional poly(A) residues], as its size was consistent with a single 2.8-kilobase (kb) glucokinase mRNA on Northern blot analysis of liver poly(A)+ RNA. The cDNA contained an open reading frame of 1392 bp that predicted a protein of 464 amino acids and a molecular mass of 52 kDa; this protein has 97% identity to rat liver glucokinase. Fourteen residues on the amino terminus of the predicted human liver glucokinase, however, differed completely from those of the predicted rat liver enzyme and could be explained by alternative splicing of a 124-bp cassette exon in human cDNA. A second glucokinase cDNA (hLGLK2), missing the 124-bp cassette exon, was isolated by PCR amplification of human liver cDNA. The hLGLK2 cDNA contained an open reading frame of 1398 bp from an ATG codon at position 164, encoding a predicted protein of 466 residues, 98% identical to the rat enzyme, but different from the predicted protein of hLGLK1 cDNA by 16 amino-terminal residues. In contrast, hLGLK1 cDNA contains multiple initiator codons upstream of the predicted initiator codon at position 294 within the cassette exon. Translation of the two mRNAs in vitro by a reticulocyte lysate system resulted in proteins of the expected size (52 kDa) for both mRNAs; yet hLGLK2 mRNA was translated four to six times more efficiently. These results suggested that the alternative splicing of a cassette exon in hLGLK1 resulted in an mRNA with an upstream initiator codon and reduced function. The relative biological activity of the two isoforms of human glucokinase and their possible developmental and/or metabolic regulation remain to be determined.
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PMID:Human liver glucokinase gene: cloning and sequence determination of two alternatively spliced cDNAs. 187 Nov 35

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl-CoA were synergistic with that by N-acetyl-glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl-CoA were competitive with each other, indicating that these two inhibitors bind to the same site. The regulatory protein exerted a non-competitive inhibition with respect to Mg-ATP at concentrations of this nucleotide less than 0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6-phosphate. The following anions inhibited glucokinase non-competitively with respect to glucose: Pi, sulfate, I-, Br-, No3-, Cl-, F- and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6-phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I- greater than Br- greater than NO3- greater than Cl- greater than F- greater than acetate and their effect was non-competitive with respect to fructose 6-phosphate. Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl-CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low-Km specific glucokinase from Bacillus stearothermophilus.
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PMID:Competitive inhibition of liver glucokinase by its regulatory protein. 188 17

In the absence of fructose 6-phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15-fold) lower potency than in the presence of a saturating concentration of fructose 6-phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6-phosphate. In the presence of regulatory protein, sorbitol 6-phosphate, a potent analog of fructose 6-phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6-phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1-phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half-maximal effect increased linearly with the concentrations of sorbitol 6-phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase. Sorbitol 6-phosphate, 2-deoxysorbitol 6-phosphate and mannitol 1-phosphate, all analogs of the open-chain configuration of fructose 6-phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6-phosphate, whereas fixed analogs of the furanose form of fructose 6-phosphate were inactive or behaved as activators. This indicated that fructose 6-phosphate in its open-chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1-phosphate, psicose 1-phosphate, ribitol 5-phosphate, analogs of fructose 1-phosphate and of ribitol 5-phosphate and, at much higher concentrations, inorganic phosphate.
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PMID:Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation. 188 18


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