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PubMed1996

Alteration of enzyme function of the type II hexokinase C-terminal half on replacements of restricted regions by corresponding regions of glucokinase.

To know the structural properties responsible for the enzymic activity of the 50-kDa C-terminal half of type II hexokinase (HKII-C) derived from rat hepatoma cell line AH130, we constructed cDNAs of HKII-C and its recombinants in which restricted regions containing highly conserved sequences, referred to as regions 2 and 3, were replaced by the corresponding regions of glucokinase. The binding domains of ATP and glucose were proposed to exist in these regions, respectively. Then, the HKII-C and chimera HKII-Cs were overexpressed in Escherichia coli BL21(DE3)pLysS. They all exhibited hexokinase activity, and their activities were inhibited by glucose-6-phosphate (Glc-6-P) competitively for ATP and uncompetitively for glucose. The replacement of region 2 of HKII-C by the corresponding region of glucokinase increased the affinity for glucose and decreased the affinity for Glc-6-P, but it did not significantly affect the affinity for ATP. In contrast, the replacement of region 3 did not cause an appreciable change in hexokinase activity. These findings suggest that region 2 is associated with the binding of ATP and Glc-6-P, and that the latter binding site is located close to the ATP binding site. In addition, region 2 was suggested to be directly related with the binding of glucose and other hexoses.

Amino Acid Sequence↗
PubMed1996

Analysis of the signaling pathway involved in the regulation of hexokinase II gene transcription by insulin.

The hexokinases, by converting glucose to glucose 6-phosphate, help maintain the glucose concentration gradient that results in the movement of glucose into cells through the facilitative glucose transporters. Hexokinase II (HKII) is the major hexokinase isoform in skeletal muscle, heart, and adipose tissue. Insulin induces HKII gene transcription in L6 myotubes, and this, in turn, increases HKII mRNA and the rates of HKII protein synthesis and glucose phosphorylation in these cells. Inhibitors of distinct insulin signaling pathways were used to dissect the molecular mechanism by which HKII gene expression is induced by insulin in L6 myotubes. Treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), or with rapamycin, an inhibitor of the pathway from the insulin receptor to p70/p85 ribosomal S6 protein kinase (p70(s6k)), prevented the induction of HKII mRNA by insulin. In contrast, treatment with PD98059, an inhibitor of mitogen-activated protein kinase activation, had no effect on insulin-induced HKII mRNA. In addition, rapamycin blocked the insulin-induced expression of an HKII promoter-chloramphenicol acetyltransferase fusion gene transiently transfected into L6 myotubes, whereas PD98059 had no such effect. These results suggest that a phosphatidylinositol 3-kinase/p70(s6k)-dependent pathway is required for regulation of HKII gene transcription by insulin and that the Ras-mitogen-activated protein kinase-dependent pathway is probably not involved.

Adipose Tissue↗
PubMed1996

Identification and characterization of basal and cyclic AMP response elements in the promoter of the rat hexokinase II gene.

Hexokinases catalyze the phosphorylation of glucose and initiate cellular glucose metabolism. Hexokinase II (HKII) is the principal hexokinase isoform in skeletal muscle, heart, and adipose tissue. Isoproterenol and exogenous cyclic AMP (cAMP) increase HKII gene transcription in L6 myotubes. Various segments of the HKII promoter that direct the expression of the chloramphenicol acetyltransferase reporter gene were transfected into L6 myotubes to identify basal and cAMP response elements. The 5'-flanking region that extends 90 base pairs upstream of the transcription start site includes a CCAAT box and a cAMP response element (CRE); both contribute to basal promoter activity and each provides an independent, maximal response to cAMP. An inverted CCAAT motif, or Y box, located just upstream of the CCAAT box, contributes to basal promoter activity but is not involved in the cAMP response. Homo- and heterodimers composed of the CRE-binding protein and activating transcription factor-1 bind specifically to the CRE. The Y box and the CCAAT box specifically bind the factor NF-Y (also known as CBF).

Animals↗
PubMed1996

Differential metabolic effects of adenovirus-mediated glucokinase and hexokinase I overexpression in rat primary hepatocytes.

The first step of glucose metabolism is the phosphorylation of glucose, catalyzed by the hexokinase family of enzymes. To address the metabolic impact of increasing glucose phosphorylation capacity in liver, rat primary hepatocytes were treated with recombinant adenoviruses containing the cDNAs encoding either rat liver glucokinase (AdCMV-GKL) or rat hexokinase I (AdCMV-HKI). Maximal glucose phosphorylation in AdCMV-GKL- and AdCMV-HKI-treated hepatocytes was increased 7.1 +/- 1.2- and 6.3 +/- 0.8-fold, respectively, over hepatocytes treated with an adenovirus expressing beta-galactosidase. Glucose usage (measured with 3 and 20 m 2-[3H]glucose and 5-[3H]glucose) was significantly increased in AdCMV-GKL-treated cells preincubated in 1 or 25 mM glucose. Treatment of hepatocytes with AdCMV-HKI also caused enhanced glucose utilization, but the increases were smaller and were less apparent in cells preincubated in high (25 mM) glucose. AdCMV-GKL-treated hepatocytes incubated for 48 h in the presence of variable glucose concentrations had glycogen levels that were maximally 15.0 +/- 0. 6-fold greater than levels in corresponding control cells. AdCMV-HKI-treated hepatocytes incubated under similar conditions had unchanged glycogen levels relative to controls. In AdCMV-GKL-treated cells, lactate output was increased to a maximum of 3.0 +/- 0.4-fold (at 25 mM glucose), glucose oxidation was increased 3.5 +/- 0.3-fold, and triglyceride production was unchanged relative to untreated cells. Among these three parameters, only lactate production was increased in AdCMV-HKI-treated cells, and then only at low glucose concentrations. We conclude that overexpression of glucokinase has potent effects on glucose storage and utilization in hepatocytes and that these effects are not matched by overexpression of hexokinase I.

Adenoviridae↗
PubMed1999

Characterization of the rat type III hexokinase gene promoter. A functional octamer 1 motif is critical for basal promoter activity.

A 1532-base pair 5'-flanking region of the gene encoding rat type III hexokinase has been cloned and sequenced. The total sequence includes positions -1548 to -17 (A of the translational start ATG as position +1). Using luciferase reporter constructs transfected into PC12 (rat pheochromocytoma) and L2 (rat lung) cells, basal promoter activity has been associated with sequence between -182 and -89. This includes a single transcriptional start site, an adenine at position -134 identified by primer extension. Together with previously cloned cDNA sequence, this accounts for an mRNA of approximately 3.9 kilobases, found by Northern blotting of RNA from rat lung and kidney. Sequence upstream of the transcriptional start site was devoid of canonical TATA and CAAT elements. An octamer 1 (Oct-1) binding site, located between positions -166 and -159 was shown by deletion analysis and site-directed mutation to be critical for promoter activity. Nuclear extracts from PC12 cells contained a protein (or proteins) specifically binding the octamer sequence, and supershift experiments with anti-Oct-1 indicated involvement of this ubiquitously expressed transcription factor in the complex. Sequence including the Oct-1 site and immediately adjacent regions was protected from DNase I digestion in footprinting experiments with nuclear extracts from PC12 cells. Reverse transcription polymerase chain reaction indicated that levels of type III hexokinase mRNA in rat tissues increased in the order brain < liver < lung approximately kidney; immunoblotting indicated that type III hexokinase protein in these tissues increased in a similar manner.

Animals↗
PubMed2003

Onset of carbon catabolite repression in Aspergillus nidulans. Parallel involvement of hexokinase and glucokinase in sugar signaling.

The role of hexose phosphorylating enzymes in the signaling of carbon catabolite repression was investigated in the filamentous fungus Aspergillus nidulans. A d-fructose non-utilizing, hexokinase-deficient (hxkA1, formerly designated frA1) strain was utilized to obtain new mutants lacking either glucokinase (glkA4) or both hexose kinases (hxkA1/glkA4). d-Glucose and d-fructose phosphorylation is completely abolished in the double mutant, which consequently cannot grow on either sugar. The glucokinase single mutant exhibits no nutritional deficiencies. Three repressible diagnostic systems, ethanol utilization (alcA and alcR genes), xylan degradation (xlnA), and acetate catabolism (facA), were analyzed in these hexose kinase mutants at the transcript level. Transcriptional repression by d-glucose is fully retained in the two single kinase mutants, whereas the hexokinase mutant is partially derepressed for d-fructose. Thus, hexokinase A and glucokinase A compensate each other for carbon catabolite repression by d-glucose in the single mutants. In contrast, both d-glucose and d-fructose repression are severely impaired for all three diagnostic systems in the double mutant. Unlike the situation in Saccharomyces cerevisiae, the hexose phosphorylating enzymes play parallel roles in glucose repression in A. nidulans.

Acetate-CoA Ligase↗
PubMed2005

Probing the role of compartmentation of glycolysis in procyclic form Trypanosoma brucei: RNA interference studies of PEX14, hexokinase, and phosphofructokinase.

Trypanosoma brucei and related organisms contain an organelle evolutionarily related to peroxisomes that sequesters glycolysis, among other pathways. We have shown previously that disruption of protein import into this organelle, the glycosome, can be accomplished through RNA interference (RNAi)-mediated knockdown of the peroxin PEX14. Decreased PEX14 in turn leads to cell death, which, at least in the procyclic stage, can be triggered by the presence of glucose. Here we show that fructose, which is taken up and metabolized by procyclic form T. brucei, and glycerol, which interfaces with the glycosomal glycolytic pathway, are also toxic during PEX14 RNAi. Earlier computer modeling studies predicted that glycolysis would be toxic to T. brucei in the absence of glycosomal compartmentation because of the intrinsic lack of feedback regulation of the parasite hexokinase and phosphofructokinase. To further test this hypothesis, we performed double RNAi, targeting hexokinase and PEX14. Knockdown of hexokinase rescued PEX14 knockdown cells from glucose toxicity, even though glycosomal proteins continue to be mislocalized to the cytosol. Knockdown of phosphofructokinase was benign in the absence of glucose but toxic in the presence of glucose. When PEX14 and phosphofructokinase mRNAs were jointly targeted for RNAi, glycerol remained toxic to the parasites. Taken together, these data indicate that the glycosome provides significant, but not complete, protection of trypanosomes from the dangerous design of glycolysis.

Animals↗
PubMed2005

Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria.

One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.

Adenosine Triphosphate↗
PubMed1979

Preparation and study of a fluorescent sugar analog competitive inhibitor of yeast hexokinase.

The preparation of 2-deoxy-2-amino-N-(5-dimethylamino-1-naphthalene sulfonyl)-glucose (III) designed as a fluorescent competitive inhibitor of hexokinase was achieved after reacting 2-deoxy-2-aminoglucose and 1-dimethylamino-5-naphthalene sulfonyl chloride. (III) showed fluorescence excitation and emission maxima in water at 330 and 507 nm, respectively. (III) was found to competitively inhibit hexokinase and a value of Ki = 3.0 x 10(-3)M was obtained for the system hexokinase B + Mg.ATP + glucose at pH 8.4.

Binding Sites↗
PubMedDate not supplied

Investigation of red blood cell carbonic anhydrase, glucose 6-phosphate dehydrogenase, hexokinase enzyme activities, and zinc concentration in patients with hyperthyroid diseases.

We have recently reported on the activities of glucose-6-phosphate dehydrogenase (G6PD), hexokinase (HK) and carbonic anhydrase I (CA-I) and II (CA-II) isoenzymes obtained from erythrocytes of healthy subjects and untreated patients with hyperthyroid diseases. Also, erythrocyte zinc concentrations were measured. Red blood cell (RBC) zinc (Zn) concentration was measured by atomic absorption spectrophotometry. Activities of carbonic anhydrase II and I isoenzymes were determined with CO2-hydratase activity method by using selective inactivation with bromopyruvate. G6PD and HK enzyme activities were measured spectrophotometrically via absorbance change (at 340 nm) in NADPH formed as a result of the reactions catalysed by these enzymes. In statistical analysis of all these parameters, activity of CA-I was 4388 +/- 207 (EU/gHb) and 2881 +/- 869 (EU/gHb) in healthy and untreated hyperthyroid subjects, respectively. The activity values for CA-II were 5391 +/- 257 (EU/gHb) and 4688 +/- 12.6 (EU/gHb) in healthy and untreated hyperthyroid subjects. Glucose 6-phosphate dehydrogenase (G6PD) activity was 10.19 +/- 1.87 (EU/gHb) in healthy group and 4.92 +/- 2.49 (EU/gHb) in patient group. While hexokinase enzyme activity was 1.575 +/- 0.898 in healthy subjects, it was 0.651 +/- 0.418 (EU/gHb) in the patient group. While erythrocyte zinc concentration in the healthy subjects was 49.32 +/- 23.5 (mg/gHb), this concentration for patients with uncontrolled hyperthyroid diseases was significantly decreased to 29.62 +/- 4.26 (mg/gHb). As a conclusion, CA-I isoenzyme, G6PD, hexokinase activities and erythrocyte zinc concentration had decreased in untreated patients carrying hyperthyroid diseases as compared to those of the healthy subjects.

Adult↗
PubMed2002

The overexpressed hexahistidine-tagged human hexokinase type III is inhibited by D-glucose.

Inhibition by its product, glucose, is a kinetic property of hexokinase type III. In this paper, we report the overexpression in Escherichia coli of human hexokinase type III. The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end. This modification confers to the product the ability to bind the Ni2+ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups. The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent. Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg. Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose. The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Contrasting molecular population genetics of four hexokinases in Drosophila melanogaster, D. simulans and D. yakuba.

As part of a larger study contrasting patterns of variation in regulatory and nonregulatory enzymes of the central metabolic pathways we have examined the molecular variation in four uncharacterized hexokinase genes unique to muscle, fat body, and testis in Drosophila melanogaster, D. simulans, and D. yakuba. Earlier isoenzyme studies had designated these genes as Hex-A, Hex-C, and Hex-t. There are two tightly linked testes-specific genes designated here as Hex-t1 and Hex-t2. Substantial and concordant differences across species are seen in levels of both amino acid and silent polymorphism. The flight muscle form Hex-A is the most conserved followed by the fat body hexokinase Hex-C and testis-specific hexokinases Hex-t1 and Hex-t2. While constraints acting at the amino acid level are expected, the silent polymorphisms follow this pattern as well. All genes are in regions of normal recombination, therefore hitchhiking and background selection are not likely causes of interlocus differences. In D. melanogaster latitudinal clines are seen for amino acid polymorphisms at the Hex-C and Hex-t2 loci. There is evidence for accelerated amino acid substitution in Hex-t1 that has lost residues known to be associated with glucose and glucose-6-phosphate binding. D. simulans shows substantial linkage phase structuring that suggests historical population subdivision.

Adipose Tissue↗
PubMed1997

Genetic expression of hexokinase and glucose phosphate isomerase in late-stage mouse preimplantation embryos: transcription activities in glucose/phosphate-containing HTF and glucose/phosphate-free P1 media.

In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.

Animals↗
PubMed1997

Interaction with D-glucose and thermal denaturation of yeast hexokinase B: A DSC study.

DSC measurements have been performed on the monomeric form of yeast hexokinase B in the absence and presence of increasing concentrations of D-glucose. The hexokinase, in the absence of D-glucose, at both pH 8.0 and 8.5, shows reproducible calorimetric profiles characterized by the presence of two partially overlapped peaks. These can be ascribed to the presence of two structural domains in the native conformation of the enzyme, that possess different thermal stabilities and are denatured more or less independently. In the presence of saturating and increasing concentrations of D-glucose, the shape of the DSC profiles dramatically changes, since a single well-shaped peak is present. The binding of D-glucose enhances the interaction between the two lobes, as evidenced by the shrinking of the protein in overall dimensions, and gives rise to DSC profiles resembling those of a single domain protein. To deconvolve the DSC curves we considered a denaturation model consisting of two sequential steps with three macroscopic states of the protein and the binding of D-glucose only to the native state. We carried out two-dimensional nonlinear regression of the excess heat capacity surface constructed with the experimental DSC curves. This approach allows the calculation of a unique set of thermodynamic parameters characterizing both the thermal denaturation of hexokinase, and the binding equilibrium between D-glucose and the enzyme. It was found that the association constant is 9,800+/-1,500 M(-1) at pH 8.0. The binding of D-glucose is entropy-driven, since the binding enthalpy is zero. This finding is rationalized by a thermodynamic cycle for the association of two molecules in aqueous solution.

Calorimetry, Differential Scanning↗
PubMed1976

Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Unique hexokinase messenger ribonucleic acids lacking the porin-binding domain are developmentally expressed in mouse spermatogenic cells.

We have identified cDNAs representing three hexokinase mRNAs (Hk1-sa, Hk1-sb, Hk1-sc) by screening mouse spermatogenic cell cDNA libraries with a mouse hepatoma cell line hexokinase (Hk1) cDNA [Arora KK, Fanciulli M, Pederson PL. J Biol Chem 1990; 265:6481-6488]. Although all three cDNAs show 99% identity to the somatic Hk1 cDNA sequence throughout most of their coding region, they differ from this sequence at the 5' end. They contain a common spermatogenic cell-specific sequence and a sequence unique to each cDNA immediately 5' to the common domain. However, they lack the porin-binding domain (PBD) present in this region of Hk1, used for binding to a pore-forming protein in the outer mitochondrial membrane. These observations appear to support a model proposed by others for hexokinase gene evolution in mammals. In addition, we found that Hk1-sb has an internal sequence that is not present in Hk1, Hk1-sa, or Hk1-sc. Moreover, Hk1-sa and Hk1-sb transcripts are developmentally expressed in mouse spermatogenic cells. Hk1-sa mRNA is first expressed during meiosis and continues to be present in postmeiotic germ cells, while the more abundant Hk1-sb mRNA is detected only in postmeiotic germ cells. These and other findings suggest that enzymes encoded by Hk1-sa, Hk1-sb, and Hk1-sc are present only in spermatogenic cells.

Aging↗
PubMed1979

Metabolic adaptations in delayed skin flaps. Glucose utilization and hexokinase activity.

The distribution of glucose and hexokinase activity was determined in the epithelial tissue of delayed bipedicled skin flaps in guinea pigs. The periods of "delay" were 1, 3, 7, 14, or 21 days. The flap survival was maximal (100% of the flap) when the flap elevation was performed either 7 or 14 days following the "delay" procedure. When the flap elevation was performed 1, 3, or 21 days following the "delay" procedure, the result was partial necrosis. A differential distribution of epithelial glucose was found within the bipedicled flaps. The lowest glucose level (30% of normal) was at a distance of 2 to 3.5 cm from the end of the caudal pedicle during the first day after the "delay" procedure. This decreased glucose content recovered toward normal levels during the later part of the "delay" period. The bipedicled flaps exhibited increased hexokinase activity during the 3-week period of the "delay," and the responses of hexokinase activity and tissue glucose levels to the "delay" procedure were reciprocal in the caudal half of the flaps.

Animals↗
PubMed1989

The hexokinase isoenzyme PII of Saccharomyces cerevisiae ia a protein kinase.

The HXK2 gene product has an important role in controlling carbon catabolite repression in Saccharomyces cerevisiae. We have raised specific antibodies against the hexokinase PII protein and have demonstrated that it is a 58 kDa phosphoprotein with protein kinase activity. The predicted amino acid sequence of the HXK2 gene product has significant homology to the conserved catalytic domain of mammalian and yeast protein kinases. Protein kinase activity was located in a different domain of the protein from the hexose-phosphorylating activity. The hexokinase PII protein level remained unchanged in P2T22D mutant cells (hxk1 HXK2 glk1) growing in a complex medium with glucose. The protein kinase activity of hexokinase PII is regulated by the glucose concentration of the culture medium. Exit from the carbon catabolite repression phase and entry into derepression phase may be controlled, in part, by modulation of the 58 kDa protein kinase activity by changes in cyclic AMP concentration.

Amino Acid Sequence↗