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 gene encoding human glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a major component of glucose sensing in pancreatic islet beta-cells, was isolated and characterized. The gene was shown by Southern blotting to exist as a single copy in the genome which mapped to chromosome 7p. It contained 12 exons including two tissue-specific first exons, one active in islet beta-cells (1B), and the other active in liver (1H), and one optional cassette exon which was expressed as a minor form in the liver. Thus the three previously reported isoforms of glucokinase mRNA were the result of tissue-specific activation of separate liver and islet promoters and subsequent alternative splicing events. Eleven exons, including 1H and the optional cassette exon 2A, were scattered over 16 kilobase (kb) in the genome, while exon 1B was separated from the rest by at least 20 kb. Although the islet promoter was found to lack a TATA box, a major transcript from the islet promoter was mapped 486 nucleotides upstream of the translation initiation site. The presence in the islet glucokinase promoter of the potential control element GCCACCAG, a homology of the regulatory element present in both human insulin (GCCACCGG) and rat insulin (GCCATCTG) genes, implied a possible tissue-specific regulatory role of this element. The liver promoter was found to contain a TATA box-like sequence, and transcription was initiated predominantly at 168 nucleotides upstream of the translation initiation site of the major isoform. A new highly polymorphic microsatellite, composed of a compound imperfect dinucleotide repeat [GT]15[GA]8CA[GA]7CA[GA]3AA[GA]2, was mapped 6 kb upstream of islet exon 1. A polymerase chain reaction-based assay was developed, and seven different sized alleles were identified in American Blacks. The sequence information reported here, along with the new polymorphic marker, will make it possible to clarify the molecular basis of potential glucokinase defects in noninsulin-dependent diabetes mellitus patients and may further elucidate the nature of genetic susceptibility to the development of this common metabolic disease.
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PMID:Human glucokinase gene: isolation, structural characterization, and identification of a microsatellite repeat polymorphism. 135 40

Using direct and competitive epitope mapping methods, 23 monoclonal antibodies (Mabs) against rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were divided into nine groups, each recognizing epitopes within defined surface regions of the N- or C-terminal domains; the latter have been associated with regulatory or catalytic functions, respectively. Reactivity of Mabs with the isolated domains was also studied. Based on the effect of various ligands on immunoreactivity, specific regions involved in ligand-induced conformational changes were identified. Adjacent epitopic regions, designated Regions F and G and located in the N- and C-terminal domains, respectively, were selectively affected by inhibitory hexose 6-phosphates (or analogs), marking these regions as being involved in transmission of the conformational signal from the regulatory N-terminal domain to the catalytic C-terminal domain. Consistent with this, the Ki for inhibition of the enzyme by the glucose 6-phosphate analog, 1,5-anhydroglucitol-6-phosphate, was markedly increased by Mabs binding in these regions, but unaffected by Mabs binding elsewhere in the molecule. Reactivity with Mabs recognizing conformationally sensitive epitopes in Region H of the C-terminal domain was greatly decreased by binding of substrate hexoses that induce closure of a cleft in the catalytic domain; selective recognition of the "open cleft" conformation, thereby preventing closure of the cleft required for progression of the catalytic cycle, can account for the marked decrease in Vmax that results from binding of these Mabs. Reactivity with Mabs binding to Region H was also decreased in the presence of inhibitory hexose 6-phosphates, implying that cleft closure was also induced by the latter; this is consistent with the suggestion that limitation of access to the C-terminal ATP binding site, resulting from cleft closure, is a factor in inhibition of the enzyme.
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PMID:Epitopic regions recognized by monoclonal antibodies against rat brain hexokinase: association with catalytic and regulatory function. 137 Jan 31

Previous work led to the conclusion that, during oxidative phosphorylation, mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from rat brain was dependent on intramitochondrially compartmented ATP as substrate. The present study demonstrated that, when oxidative phosphorylation was functioning concurrently, mitochondrial creatine kinase could also generate intramitochondrial ATP serving as substrate for hexokinase. In the absence of concurrent oxidative phosphorylation, the kinetics of glucose phosphorylation with ATP generated by creatine kinase were not consistent with the supply of ATP from a saturable intramitochondrial compartment as formed during oxidative phosphorylation. Evidence for intramitochondrially compartmented ATP, generated by creatine kinase, was obtained; this was distinct from compartmented ATP generated by oxidative phosphorylation in terms of kinetics of generation of the compartment and its capacity, sensitivity to release by carboxyatractyloside, and sensitivity to disruption by digitonin. That oxidative phosphorylation did induce a dependence on intramitochondrial ATP as a substrate was further indicated by the observation that, although the initial rate of glucose phosphorylation by mitochondrial hexokinase depended on the extramitochondrial concentration of ATP present at the time oxidative phosphorylation was initiated, a final steady state rate of glucose phosphorylation was attained that was independent of extramitochondrial ATP levels. These and previous results emphasize the probable importance of nucleotide compartmentation in regulation of cerebral glycolytic and oxidative metabolism.
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PMID:Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase. 144 44

Despite the extensive sequence similarity between the N- and C-terminal halves of the 100-kDa molecular weight mammalian hexokinases (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), reflecting their evolutionary origin by duplication and fusion of a gene coding for a smaller ancestral hexokinase, there is evidence for a functional division, with the C-terminal domain retaining a catalytic role while the N-terminal domain serves a regulatory function [binding of the product inhibitor, glucose 6-phosphate) (Glc-G-P)]. Conversion of Ser603 to Ala in the C-terminal domain of rat Type I hexokinase, expressed in COS-1 cells, resulted in drastic reduction of catalytic activity; Ser603 is analogous to Ser158, a residue of critical catalytic importance in the homologous yeast hexokinase. In contrast, conversion of Ser155 to Ala in the N-terminal domain (analogous to Ser603 in the C-terminal domain) of rat Type I hexokinase had no effect on catalytic activity or on inhibition of the enzyme by the Glc-6-P analog, 1,5-anhydroglucitol-6-P. Immunoreactivity with monoclonal antibodies recognizing conformationally sensitive epitopes was not affected, indicating that neither mutation resulted in gross structural perturbation. These results are consistent with the assignment of catalytic function, involving Ser603, to the C-terminal domain, and demonstrate that the analogous Ser155 is not critical for either catalytic or regulatory function. The Type I isozyme, expressed in COS-1 cells, retained the ability to bind to mitochondria in a Glc-6-P-sensitive manner, as previously found with the enzyme isolated from mammalian tissues.
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PMID:Functional consequences of mutation of highly conserved serine residues, found at equivalent positions in the N- and C-terminal domains of mammalian hexokinases. 152 37

The distribution of the type III isozyme of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in rat kidney, liver, spleen, lung, and brain was determined immunohistochemically, using a monoclonal antibody generated against the enzyme purified from rat Novikoff hepatoma. In all tissues, specific cell types exhibited intense staining at the nuclear periphery, as confirmed by analysis using confocal microscopy. Isolated nuclei from kidney or liver were devoid of detectable type III hexokinase, although the enzyme was found in the "soluble" fraction from kidney or liver homogenates; these results suggest that the type III isozyme is associated in a labile manner with the external surface of the nucleus, with this association being disrupted by conventional homogenization and nuclear isolation procedures. The nuclear localization of the type III isozyme contrasts with previously demonstrated association of the type I and II isozymes with mitochondria. The physiological significance of a nuclear localization for the type III isozyme remains unclear. However, it was noted that many of the cells exhibiting prominent nuclear staining for type III hexokinase are endothelial or epithelial cells, suggesting a possible relationship between nuclear type III hexokinase and transport functions which are prominent in such cells.
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PMID:Localization of the type III isozyme of hexokinase at the nuclear periphery. 156 4

Two partial-length cDNAs encoding the type 1 human hexokinase (ATP:D-hexose 6-phosphotransferase) were isolated from a placenta cDNA library using a 50-bp oligonucleotide synthesized according to the known sequence of human HK1. Using the larger (1.8 kb) cDNA insert as a probe and a panel of human-hamster somatic cell hybrids, we were able to assign the HK1 gene to the long arm of chromosome 10.
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PMID:Mapping of human hexokinase 1 gene to 10q11----qter. 157 68

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) of rat brain mitochondria is associated with membrane regions thought to correspond to contact sites (regions of close interaction of the inner and outer mitochondrial membranes). Two intramitochondrial compartments of ATP also appear to be located at contact sites, and are dependent on oxidative phosphorylation for their generation. Neither of these compartments was associated with the intermembranal space containing adenylate kinase, nor was there detectable intramitochondrial compartmentation of ATP generated by the adenylate kinase reaction. Formation of these compartments was not dependent on the presence of bound hexokinase since equivalent amounts of compartmented ATP were found in mitochondria from which a major portion of the hexokinase had been removed by treatment with Glc-6-P. During active oxidative phosphorylation, mitochondrially bound hexokinase is totally dependent upon intramitochondrially compartmented ATP as a substrate. Both the levels of ATP in the intramitochondrial compartments and the rate of glucose phosphorylation by mitochondrially bound hexokinase were shown to be correlated with the rate of oxidative phosphorylation. This dependence of hexokinase on intramitochondrial ATP levels that reflect the status of mitochondrial oxidative metabolism provides a mechanism by which hexokinase can serve as a mediator, coordinating the rate at which glucose is introduced into the glycolytic pathway with terminal oxidative stages of metabolism and avoiding the accumulation of lactate which has been associated with toxic effects on the brain.
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PMID:Coordinated regulation of cerebral glycolytic and oxidative metabolism, mediated by mitochondrially bound hexokinase dependent on intramitochondrially generated ATP. 163 53

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been studied by differential scanning calorimetry. In "high-ionic-strength" buffer (50 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), and assuming two-state behavior with calorimetric enthalpy equal to van't Hoff enthalpy, the endotherm could be deconvoluted into two transitions with Tm values of about 48 and 51 degrees C and enthalpies of about 109 and 112 kcal/mol, respectively. A similar endotherm was seen when glucose or glucose 6-phosphate was present, except that Tm values for both transitions were increased. The glucose analog, N-acetylglucosamine, had no observable effect on the endotherm, which is in agreement with previous studies indicating that this ligand, unlike glucose and glucose 6-phosphate, does not induce conformational changes that lead to increased stability of the enzyme. In "low-ionic-strength" buffer (5 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), the transitions were partially resolved even in the absence of ligands, with Tm values of about 49 and 55 degrees C. Due to difficulties with erratic baseline behavior under the low-ionic-strength conditions, enthalpies were not routinely determined, but these appeared to be similar to those seen in high-ionic-strength buffer. Also similar was the increase in stability, as reflected by the increase in Tm for both transitions, when glucose or glucose 6-phosphate was present. Correlation of these transitions with specific regions of the molecule was established by analysis of enzyme in which the domain corresponding to the first transition was selectively denatured by a partial scan in the calorimeter. Subsequent rescanning of these samples showed only the second transition, confirming the selective denaturation of the domain corresponding to the first transition and retention of the folded structure of the second domain. Discrimination between denatured (first transition) and undenatured (second transition) domains was based on the markedly increased susceptibility of the denatured region to tryptic digestion; regions of the molecule that retained their folded structure and resistance to proteolysis were identified by immunoblotting techniques using monoclonal antibodies recognizing epitopes having defined locations within the overall sequence. Based on this analysis, the first transition corresponds to unfolding of the C-terminal half of the molecule, with the second transition resulting from unfolding of the more stable N-terminal half. The order of unfolding could be reversed in the presence of ATP-Mg2+ and N-acetylglucosamine, conditions which have been shown to result in selective stabilization of the C-terminal domain.
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PMID:Differential scanning calorimetric study of rat brain hexokinase: domain structure and stability. 168 63

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

Clones containing cDNA coding for the Type III isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were isolated from a library prepared in lambda gt10 with rat liver mRNA. Three clones were characterized. Their composite sequence includes the entire coding region for Type III hexokinase, 3' untranslated sequence extending into the polyadenylated region, and 80 bp of 5' untranslated sequence. Extensive similarity in sequence of N- and C-terminal halves of the enzyme, previously seen with the Type I isozyme, is consistent with the view that these 100-kDa mammalian hexokinases are the evolutionary result of duplication and fusion of a gene coding for an ancestral hexokinase having a molecular weight of approximately 50 kDa. Extensive similarities are seen between sequences of the Type I and III isozymes, and those reported for mammalian glucokinase (also called Type IV hexokinase) and for the hexokinase and glucokinase of yeast. Residues thought to be involved in catalytic function are highly conserved in all of these enzymes. Based on a quantitative comparison of sequence similarities, it is concluded that the 50-kDa mammalian glucokinase is more closely related to the 100-kDa mammalian enzymes than it is to the 50-kDa enzymes from yeast. One interpretation of this might be that the mammalian glucokinase arose by resplitting of the gene coding for the 100-kDa mammalian hexokinases.
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PMID:Complete amino acid sequence of the type III isozyme of rat hexokinase, deduced from the cloned cDNA. 189 38


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