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At least 811 records · Page 45Linked to original sources
PubMed1986

Mechanism of inactivation of hexokinase PII of Saccharomyces cerevisiae by D-xylose.

The mechanism of inactivation of hexokinase PII of Saccharomyces cerevisiae by D-xylose was characterized. Inactivation was dependent on the presence of MgATP and was irreversible. Inactivation involved phosphorylation of the protein. Observation of the carbon catabolite repression of selected enzymes showed that invertase and maltase synthesis were not repressed when hexokinase PII was phosphorylated.

Adenosine Triphosphate↗
PubMed1997

Hexokinase as a sugar sensor in higher plants.

The mechanisms by which higher plants recognize and respond to sugars are largely unknown. Here, we present evidence that the first enzyme in the hexose assimilation pathway, hexokinase (HXK), acts as a sensor for plant sugar responses. Transgenic Arabidopsis plants expressing antisense hexokinase (AtHXK) genes are sugar hyposensitive, whereas plants overexpressing AtHXK are sugar hypersensitive. The transgenic plants exhibited a wide spectrum of altered sugar responses in seedling development and in gene activation and repression. Furthermore, overexpressing the yeast sugar sensor YHXK2 caused a dominant negative effect by elevating HXK catalytic activity but reducing sugar sensitivity in transgenic plants. The result suggests that HXK is a dual-function enzyme with a distinct regulatory function not interchangeable between plants and yeast.

Amino Acid Sequence↗
PubMed1975

Heart hexokinase: quaternary structure changes accompanying the binding of regulatory molecules.

Heart hexokinase monomer has a molecular weight of 97000 and so20,w 5.2 S. It exists in equilibrium with dimer of 194000 molecular weight and so20,w 8.1 S. The proportions of monomer and dimer presence of added ligands are 91% and 9% respectively. The existence of these forms may be demonstrated by separation on electrophoresis or chromatography. In the presence of the regulatory molecule glucose 6-phosphate, the dimer form of the enzyme is favoured. The glucose 6-phosphate mediated dimerisation is abolished in the presence of phosphate or ATP-Mg and less effectively by free ATP. Glucose has no effect on the manomer-dimer equilibrium. On prolonged storage of hexokinase in glucose 6-phosphate polymers are also formed and polymerisation is further enhanced by removal of the ligand.

Adenosine Triphosphate↗
PubMed1976

Studies on the active site of yeast hexokinase. Specific phosphorylation of a serine residue induced by D-xylose and ATPMg.

Yeast hexokinase A(ATP:D-hexose 6-phosphotransferase) is inactivated when incubated in the presence of xylose and ATPMg, or in the presence of D-lyxose in a reaction medium in which ATPMg is being continuously regenerated (phosphoenolpyruvate and pyruvate kinase). The inactivation is due to the phorphorylation of the protein. A linear relationship was observed between the inactivation and the incorporation of 32P from [gamma-32P] ATP. All hexokinase and ATPase activity of the enzyme is lost when one phosphoryl group is incorporated per enzyme subunit (molecular weight 51,000). The phosphoryl group is covalently bound by a ester linkage with a serine residue of the protein.

Adenosine Triphosphate↗
PubMed1977

Chemical studies on yeast hexokinase. Specific modification of a single tyrosyl residue with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

1. Of the 15 tyrosyl residues/subunit of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase) only one residue is specifically modified at pH 8.0 with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. 2. The acylation of this single tyrosyl residue leads to the loss of the enzyme activities (hexokinase and ATPase) by a first-order process, which can be fully reversed by treatment with hydroxylamine. 3. ATP does not protect the enzyme against chemical modification and inactivation; however, glucose exerts a noticeable though indirect protection effect against chemical modification and inactivation. 4. The chemically modified enzyme, purified by column chromatography, has 14% of the activity of the native enzyme, but the Km for ATP-Mg or glucose remains unchanged as does the pH optimum of activity. Results of conformational studies (ultracentrifugation, fluorescence, thermostability and chemical reactivity of the sulfhydryl groups) indicate that the decrease of enzyme activity due to the modification of the tyrosyl residue is related to a localized perturbation of the enzyme active-center region.

Carbodiimides↗
PubMed1984

Simultaneous purification of hexokinase, class-I fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase from Trypanosoma brucei.

A method is presented for the simultaneous purification of hexokinase, fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase, and the partial purification of glycerol-3-phosphate dehydrogenase (NAD+), 6-phosphofructokinase, glucosephosphate isomerase, and glycerol kinase from Trypanosoma brucei. As a first step, the glycosomes, microbody-like organelles of Trypanosomatidae, containing almost exclusively enzymes involved in glucose and glycerol metabolism [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360-364], were purified eightfold from homogenates with an average yield of 38%. Subsequently, the glycosomal content was subjected to hydrophobic interaction chromatography on phenyl-Sepharose. This step results in pure hexokinase (15% final yield) and almost pure triosephosphate isomerase, while the other glycosomal enzymes elute as mixtures of two or three enzymes. Triosephosphate isomerase was further purified to homogeneity on CM-cellulose (33% final yield), while phosphoglycerate kinase and fructose-bisphosphate aldolase were separated from each other and purified to homogeneity by affinity chromatography using ATP-Sepharose (25% and 30% final yields, respectively). Fructose-bisphosphate aldolase was further characterized as a typical class I enzyme.

Animals↗
PubMed1986

Mechanistic origin of the kinetic cooperativity of hexokinase type L1 from wheat germ.

A theoretical analysis is presented which shows that initial velocity data for hexokinase L1 catalysis of glucose phosphorylation by MgATP cannot be reconciled with the observed rate of the 'mnemonical' conformational transition which has been proposed to account for the kinetic cooperativity of the enzyme. The basic kinetic properties of hexokinase L1 and other allegedly 'mnemonical' enzymes appear to be fully consistent with an ordered ternary-complex mechanism in which the leading substrate participates in abortive-complex formation. It is concluded that, so far, no enzyme displaying kinetic cooperativity has been convincingly demonstrated to operate by a 'mnemonical' type of reaction mechanism.

Allosteric Regulation↗
PubMed1996

Cloning and biochemical characterisation of an Aspergillus niger glucokinase. Evidence for the presence of separate glucokinase and hexokinase enzymes.

The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of the gene was determined, and the deduced amino acid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a pI of 5.2. The enzyme has high affinity for glucose (K(m) 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K(m) 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non-competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The kcal (turnover number) decreases rapidly below pH 7.5 (56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity. In addition, proof is provided for the presence of a second hexosephosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6-phosphate.

Adenosine Diphosphate↗
PubMed1975

Genetically determined polymorphism of a form of hexokinase, HK III, found in human leucocytes.

1. A form of hexokinase which appears to correspond to HK III, seen in liver, spleen and lung, has been identified in human polymorphonuclear leucocytes. HK III activity has not been demonstrated in lymphocytes, permanent lymphoblastoid cell lines or in cultured fibroblasts. 2. In fresh leucocyte preparations HK III usually appeared as a single band, but in extracts of post-mortem tissues multibanded patterns were seen. Leucocyte preparations also give multi-banded patterns after storage for more than a few hours. 3. In certain individuals the HK III showed a two-banded pattern even in fresh preparations. Repeat samples and family studies suggested that this pattern was genetically determined and represented a heterozygote for a common allele HK1III and a less common variant HK2III. Out of a total of 330 English people successfully tested 10 probable heterozygotes were found. 4. It is suggested that HK III may be a monomeric enzyme whose genetic determination is independent of that of the other main forms of hexokinase.

Alleles↗
PubMed1983

Retinal hexokinase: kinetic properties and the effect of cyclic 3',5'-adenosine monophosphate.

We measured hexokinase (EC 2.7.1.1) activity in particulate and soluble fractions isolated from bullfrog (Rana catesbeiana) retinas. Seventy-three percent of the hexokinase (HK) activity was associated with the particulate fraction, 27% with the soluble fraction. Both HK fractions could phosphorylate fructose, glucose, 2-deoxy-D-glucose, and mannose, but not galactose. The Km for glucose was 0.14 mM, for 2-deoxy-D-glucose, 3.6 mM. With glucose as substrate, the Vmax for particulate HK was 125-148 microM retina-1 min-1, for soluble HK, 37 microM retina-1 min-1. Product inhibition of particulate HK activity by glucose 6-phosphate was marked, whereas 2-deoxy-D-glucose 6-phosphate did not inhibit the activity. Cyclic AMP stimulated the HK activity of both retinal fractions nearly twofold at concentrations of 0.2-0.8 mM; AMP was much less effective in this regard.

Animals↗
PubMed1986

Correlation of hexokinase content and basal energy metabolism in discrete regions of rat brain.

Brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) levels in seven regions of rat brain were estimated by photometric measurement of immunofluorescence in cryostat-cut sections. When compared with basal rates of glucose metabolism in these regions, estimated by the 6-[14C]glucose method, a significant correlation was observed. Thus, hexokinase content reflects metabolic energy demands.

Animals↗
PubMed1975

Kinetic properties of hexokinase in resected temporal lobes of patients with drug-resistant epilepsy.

Properties of the cerebral glycolytic enzyme, hexokinase, were studied in biopsy samples of human temporal lobe, obtained during lobectomy for drug-resistant epilepsy and compared "blind" with contol biopsy samples of human cerebral cortex. No significant changes in the total activity or subcellular distribution of the enzyme were observed but the Km value for glucose was altered. The 17 control samples gave a normal mean value for Km (glucose) of 0.05 mM and the 14 epileptic samples gave a significantly higher mean value of 0.09 mM. The drugs used in previous treatment of the epilepsies were "scored" with respect to type and dose; analysis of these in relation to the kinetic results eliminated the possibility that the increase in Km value was an artifact due to the drugs. The observed change in enzyme kinetic properties is discussed in terms of potential interactions of small molecules with the isoenzymes of cerebral hexokinase.

Age Factors↗
PubMed1968

Hexokinase isoenzymes in human erythrocytes.

The electrophoretic mobility of hexokinase from human erythrocytes and other tissues was studied with a new method that depends on the fluorescence of reduced nicotinamide-adenine dinucleotide phosphate for detecting enzyme activity on starch gel. The hexokinase of cord-blood erythrocytes has slightly different electrophoretic properties from that of adult red cells. Type I enzyme is split into type I(A) and type I(F); the latter is more intense in cord blood; in hemolyzates of adult blood, the activity of the two bands is usually about equal. No type II enzyme was found in cord blood. The double type I band was present in red cells from adult rabbits.

Animals↗
PubMed1983

Pleiotropic mutations regulating resistance to glucose repression in Saccharomyces carlsbergensis are allelic to the structural gene for hexokinase B.

Previously, we described a mutation glr1-1 in Saccharomyces carlsbergensis which pleiotropically relieves the synthesis of the following enzymes from glucose repression: maltase, galactokinase, alpha-galactosidase, NADH:cytochrome c reductase, and cytochrome c oxidase (C. A. Michels and A. Romanowski, J. Bacteriol, 143:674-679, 1980.) In this report, we demonstrate that glr1-1 and two other alleles, glr1-3 and glr1-16, are also insensitive to the glucose repression of invertase synthesis. Determinations of the levels of hexokinase activity and the rate of glucose transport in these mutants show that both are reduced as compared with the parent strain. Complementation tests and genetic analysis indicate that the glr1 mutations are allelic to HXK2, the structural gene for hexokinase B. The significance of this result is discussed with regard to the mechanism of glucose repression in S. carlsbergensis.

Alleles↗
PubMed2005

Characterization of the RokA and HexA broad-substrate-specificity hexokinases from Bacteroides fragilis and their role in hexose and N-acetylglucosamine utilization.

Bacteroides fragilis, a human gastrointestinal commensal and an opportunistic pathogen, utilizes simple and complex sugars and polysaccharides for growth in the large intestine and at sites of infection. Because B. fragilis lacks transport-linked sugar phosphorylation systems, cytoplasmic kinase(s) was expected to be required for the phosphorylation of hexoses and hexosamines. We have now identified two hexose kinases that are important for growth of B. fragilis on glucose, mannose, and other sugars. One kinase (RokA), a member of the ROK family of proteins, was found to be the sole kinase for activation of N-acetyl-D-glucosamine (NAG). The other kinase (HexA) is responsible for the majority of the glucose kinase activity in the cell, although a hexA deletion mutant strain was not defective for growth on any substrate tested. Deletion of both the rokA and hexA kinase genes resulted in inability of the cell to use glucose, mannose, NAG, and many other sugars. We purified RokA and determined its approximate molecular mass to be 36.5 kDa. The purified RokA protein was shown to phosphorylate several substrates, including glucose, NAG, and mannose, but not N-acetylmannosamine or N-acetylneuraminic acid. Phylogenetic analysis of RokA showed that it is most similar to kinases from the Cytophaga-Flavibacterium-Bacteroides group, while HexA was most similar to other bacterial hexokinases and eukaryotic hexokinases.

Acetylglucosamine↗
PubMed1988

A new metabolic muscle disease due to abnormal hexokinase activity.

A 34 year old man presented with an 8 year history of mild muscle pain and stiffness on exertion especially in the cold. Clinical examination was normal. Apart from a mild persistent leucocytosis, his routine investigations were normal including creatine kinase activity, electromyography and nerve conduction studies. An ischaemic exercise test produced a slow and incomplete rise in lactate. Histological examination showed non-specific myopathic changes in some quadriceps femoris muscle fibres. Investigation of muscle metabolism by spectrofluorometric analysis of muscle enzyme activity and by muscle fibre incubation studies revealed a severe defect in glucose phosphorylation, associated with an electrophoretically abnormal hexokinase. Further metabolic studies suggest that the block in glucose metabolism is by-passed via an enhanced phosphorylation of fructose by the abnormal hexokinase.

Adult↗