Purification and properties of the cytoplasmic hexokinase from rabbit brain

About 90% of the total hexokinase activity in rabbit brain was found to be associated with mitochondria while the remaining part was found in the cytosolic fraction. The soluble enzyme was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The molecular weight of the purified hexokinase was found to be approximately 98,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.2 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses. Mg . ATP2-, among the nucleotide substrates, was the most effective phosphate donor. The properties of the purified cytoplasmatic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in molecular weight, isoelectric point, pH dependence of activity, electrophoretic mobility and affinity for glucose and Mg.ATP2-. However, the temperature dependence of activity, and the specificity for several hexose substrates were markedly different.     

Sugar repression in the methylotrophic yeast Hansenula polymorpha studied by using hexokinase-negative, glucokinase-negative and double kinase-negative mutants

Two glucose-phosphorylating enzymes, a hexokinase phosphorylating both glucose and fructose, and a glucose-specific glucokinase were electrophoretically separated in the methylotrophic yeastHansenula polymorpha. Hexokinase-negative mutants were isolated inH. polymorpha by using mutagenesis, selection and genetic crosses. Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was studied in different hexose kinase mutants. In the wild type and in mutants possessing either hexokinase or glucokinase, glucose repressed the synthesis of maltase, alcohol oxidase and catalase. Glucose repression of alcohol oxidase and catalase was abolished in mutants lacking both glucose-phosphorylating enzymes (i.e. in double kinase-negative mutants). Thus, glucose repression inH. polymorpha cells requires a glucose-phosphorylating enzyme, either hexokinase or glucokinase. The presence of fructose-phosphorylating hexokinase in the cell was specifically needed for fructose repression of alcohol oxidase, catalase and maltase. Hence, glucose or fructose has to be phosphorylated in order to cause repression of the synthesis of these enzymes inH. polymorpha suggesting that sugar repression in this yeast therefore relies on the catalytic activity of hexose kinases.     

A pH sensitive colorometric assay for the high-throughput screening of enzyme inhibitors and substrates: a case study using kinases

We have developed an uncoupled, pH sensitive kinase assay that can be used for high-throughput screening of potential inhibitors or for determining substrate specificity. Kinases catalyze the transfer of a gamma-phosphoryl group from ATP to an appropriate hydroxyl acceptor with the release of a proton. This assay is based on the detection of this proton using an appropriately matched buffer/indicator system. The assay was used to measure the activity of four readily available kinases, including hexokinase, glucokinase, glycerokinase, and pyruvate kinase, which was run in the reverse direction. We also went on to screen a small series of mono- and diphosphonucleotides for inhibition of hexokinase as well as a modest set of potential hexokinase substrates. We determined sucrose to be a modest substrate for hexokinase with a K(m) of 1.8 +/- 0.2 mM, a k(cat) of 142 +/- 3 min(-1), and a V(max) that is 15% of that for glucose. Given the importance of kinases in a diverse array of biological functions and disease states, there is a need for a simple, rapid assay system. We feel this assay will lend itself well to meet this end. This method should be applicable to many other enzymatic reactions which involve a change in pH.     

Glucokinase and fructokinase of Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus contain glucokinase and not a hexokinase of broad hexose specificity. Tritrichomonas foetus also contains a specific fructokinase which could be resolved from glucokinase by anion exchange chromatography. Native T. vaginalis glucokinase had a Mr of 76,000, and SDS-PAG electrophoresis showed two equally stained bands corresponding to Mr 40,000 and 38,000. Glucose and ATP were by far the best substrates for both trichomonad glucokinases, with Km values as low as 33-35 microM and 75-83 microM, respectively. Substrate saturation curves for these enzymes were all hyperbolic. Tritrichomonas foetus fructokinase required fructose and ATP, with Km values of 200 microM and 81 microM. None of the activities was affected by a number of potential regulatory metabolites, including glucose-6-phosphate. The only exception was AMP which in supraphysiological concentrations had an inhibitory effect on T. foetus fructokinase. In conclusion, the absence of regulation at the hexose phosphorylation step described here, as well as the presence of an easily reversible PPi: fructose-6-phosphate 1-phosphotransferase described previously (Mertens, E., Van Schaftingen, E. & Müller, M. 1989. Mol. Biochem. Parasitol., 37:183-190), suggest that the rate of the 1st part of glycolysis in trichomonads is controlled only by the intracellular availability of hexoses.     

Is glucokinase responsible for the anomeric specificity of glycolysis in pancreatic islets?

At a low concentration of D-glucose (3.3 mM), the phosphorylation rate of this hexose in rat pancreatic islet homogenates incubated at 8 degrees C is higher with the beta- than with the alpha-anomer, as expected from the anomeric specificity of hexokinase. In the presence of a high concentration of glucose 6-phosphate (3.0 mM), which inhibits hexokinase but not glucokinase, the phosphorylation rates of the two anomers are not significantly different from one another. Nevertheless, in intact islets exposed at 8 degrees C to the same low concentration of D-glucose, the alpha-anomer augments, more than the beta-anomer, the production of lactic acid and net uptake of 45Ca. At the same concentration (3.3 mM), the alpha-anomer is also more potent than the beta-anomer in enhancing insulin release from perfused pancreases stimulated at 37 degrees C by L-leucine or by the combination of Ba2+ and theophylline. It is concluded that the participation of glucokinase is not essential for the anomeric specificity of glycolysis and insulin release in rat pancreatic islets.     

Hexose kinases from Rhodotorula glutinis. Identification and properties of an hexokinase and a glucokinase.

An hexokinase (EC 2.7.1.1) and a glucokinase (EC 2.7.1.2) from the red yeast Rhodotorula glutinis are described. Both enzymes have been separated and some of their properties studied. The two enzymes share many properties, the K_mfor glucose is 0.1 mm for both enzymes and the K_m values for ATP are 0.5 mm and 0.6 mm respectively for hexokinase and glucokinase. The hexokinase shows a K_m of 2 mm for fructose and 0.1 mm for mannose; the glucokinase has a K_m for mannose of 0.2 mm. Both enzymes are constitutive, show competitive inhibition by N-acetylglucosamine and xylose, have weak affinity for glucosamine and exhibit a broad pH optimum. The molecular weights determined by gel filtration are 110,000 for glucokinase and 96,000 for hexokinase. The maximal activity of both hexose kinases nearly accounts for glucose utilization by Rh. glutinis.     

STUDIES ON THIAMINE TRIPHOSPHATE II. THIAMINE TRIPHOSPHATE AS PHOSPHATE DONOR

Yeast hexokinase, muscle fructokinase and glucokinase and liverhomogenate have no activity of transferring phosphate from TTPto hexose. A fraction precipitated from yeast autolysate at0.5–0.7 saturation of ammonium sulfate rendered TTP totransfer its terminal phosphoric residue to ADP to form ATP. (Received November 25, 1961; )     

Glucokinase and Fructokinase of Trichomonas vaginalis and Tritrichomonas foetus

ABSTRACT. Trichomonas vaginalis and Tritrichomonas foetus contain glucokinase and not a hexokinase of broad hexose specificity. Tritrichomonas foetus also contains a specific fructokinase which could be resolved from glucokinase by anion exchange chromatography. Native T. vaginalis glucokinase had a Mr of 76,000, and SDS-PAG electrophoresis showed two equally stained bands corresponding to Mr 40,000 and 38,000. Glucose and ATP were by far the best substrates for both trichomonad glucokinases, with Km values as low as 33–35 μM and 75–83 μM, respectively. Substrate saturation curves for these enzymes were all hyperbolic. Tritrichomonas foetus fructokinase required fructose and ATP, with Km values of 200 μM and 81 μM. None of the activities was affected by a number of potential regulatory metabolites, including glucose-6-phosphate. The only exception was AMP which in supraphysiological concentrations had an inhibitory effect on T. foetus fructokinase. In conclusion, the absence of regulation at the hexose phosphorylation step described here, as well as the presence of an easily reversible PPi: fructose-6-phosphate 1 -phosphotransferase described previously (Mertens, E., Van Schaftingen, E. & Müller, M. 1989. Mol. Biochem. Parasitol. , 37 :183–190), suggest that the rate of the 1st part of glycolysis in trichomonads is controlled only by the intracellular availability of hexoses.     

Crystal structures of an ATP-dependent hexokinase with broad substrate specificity from the hyperthermophilic archaeon Sulfolobus tokodaii

Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg(2+), and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg(2+) ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.     

Subcellular distribution of hexokinase isoenzymes in pancreatic islet cells exposed to digitonin after incubation at a low or high concentration of D-glucose

It was recently proposed that stimulation of pancreatic islet by D-glucose results in the translocation of glucokinase from the perinuclear area to the cell periphery, where the enzyme might conceivably interact with either the glucose transporter GLUT-2 or some other proteins and, by doing so, become better able to express its full catalytic activity. To explore the possible interaction between glucokinase and the cell boundary, dispersed rat pancreatic islet cells were preincubated for 60 min at a low (2.8 mM) or high (16.7 mM) concentration of D-glucose, then exposed for 1 min to digitonin (0.5 mg/ml) and eventually centrifuged through a layer of oil for separation of the cell pellet from the supernatant fraction containing the material released by digitonin. Under these conditions, the bulk of lactate dehydrogenase and glutamate dehydrogenase activities were recovered in the supernatant fraction and cell pellet, respectively. The measurement of hexokinase isoenzyme activities in th e two subcellular fractions, as conducted at low or high hexose concentrations and in either the absence or presence of exogenous hexose phosphates (3.0 mM glucose 6-phosphate and 1.0 mM fructose 1-phosphate) indicated a preferential location of the low-Km hexokinase in the cell pellet and of the high-Km glucokinase in the cytosolic fraction. Such a distribution pattern failed to be significantly affected by the concentration of D-glucose used during the initial incubation of the dispersed islet cells. These findings argue against the view that the glucose-induced translocation of glucokinase would result in any sizeable binding of the enzyme to a plasma membrane-associated protein. (Mol Cell Biochem 175: 131–136, 1997)     

Hexokinases of tobacco leaves: Subcellular localization and characterization

Subeellular localization of the enzymes which phosphorylate hexoses was studied in photosynthesizing tobacco leaves by means of differential centrifugation and centrifugation in sucrose gradient. More than 80 % of the total hexokinase activity of leaf tissues were found to be associated with the particulate fraction of mitochondria; however, the ratio of the particulate hexokinase fraction to the soluble fraction was influenced by the extraction medium applied. The particulate hexokinases showed a high affinity to glucose (Km = 26.8 μM) and a relatively low affinity to fructose (Km = 17.6 mM). They had a broad pH optimum, because 81 % of the phosphorylating activity obtained for glucose and 75 % of the activity obtained for fructose occurred in pH range from 7.9 to 9.1 (Tris-HCl buffer). The hexokinases were Mg2+ dependent with the highest activity occurring at equimolar Mg2+ and ATP concentrations. Their activity was enhanced by KC1, NaCl, and (NH4)2SO4 at 30 to 120 mM concentrations.     

The activity of deoxyribonucleic acid polymerase in some species of algae

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue-green algae Anabaena and Anacystis contain a 5-20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue-green algae show a pH optimum of 9 and prefer a relatively low Mg(2+) concentration (1-3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg(2+) concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5'-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA-cellulose; 6-40-fold purifications of the enzyme were obtained by chromatography on columns of DNA-cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.     

Inhibition and inactivation of glucose-phosphorylating enzymes from Saccharomyces cerevisiae by D-xylose

Three glucose-phosphorylating enzymes were separated from cell-free extracts of Saccharomyces cerevisiae by hydroxylapatite chromatography. Variations in the amounts of these enzymes in cells growing on glucose and on ethanol showed that hexokinase PI was a constitutive enzyme, whereas synthesis of hexokinase PII and glucokinase were regulated by the carbon source used. Glucokinase proved to be a glucomannokinase with Km values of 0.04 mM for both glucose and mannose. D-Xylose produced an irreversible inactivation of the three glucose-phosphorylating enzymes depending on the presence or absence of ATP. Hexokinase PI inactivation required ATP, while hexokinase PII was inactivated by D-xylose without ATP in the reaction mixture. Glucokinase was protected by ATP from this inactivation. D-Xylose acted as a competitive inhibitor of hexokinase PI and glucokinase and as a non-competitive inhibitor of hexokinase PII.     

The interaction of yeast hexokinase with Procion Green H-4G.

1. A number of reactive triazine dyes specifically and irreversibly inactive yeast hexokinase at pH 8.5 and 33 degrees C. Under these conditions, the enzyme is readily inactivated by 100 microM-Procion Green H-4G, Blue H-B, Turquoise H-7G and Turquoise H-A, is less readily inactivated by Procion Brown H-2G. Green HE-4BD, Red HE-3B and Yellow H-5G and is not inactivated at all by Procion Yellow H-A. 2. The inactivation of hexokinase by Procion Green H-4G is competitively inhibited by the adenine nucleotides ATP and ADP and the sugar substrates D-glucose, D-mannose and D-fructose but not by nonsubstrates such as D-arabinose and D-galactose. 3. Quantitatively inhibited hexokinase contains approx. 1 mol of dye per mol of monomer of mol.wt. 51000. The inhibition is irreversible and activity cannot be recovered on incubation with high concentration (20 mM) of ATP or D-glucose. 4. Mg2+ protects the enzyme against inactivation by Procion Green H-4G but enhances the rate of inactivation by all the other Procion dyes tested. In the presence of 10 mM-Mg2+ the apparent dissociation constant between enzyme and dye is reduced from 199.0 microM to 41.6 microM. Binding of the dye to hexokinase is accompanied by characteristic spectral changes in the range 560-700 nm. 5. Mg2+ promotes binding of yeast hexokinase to agarose-immobilized Procion Green H-4G but not to the other dyes tested. Elution could be effected by omission of Mg2+ from the column irrigants or by inclusion of MgATP or D-glucose, but not by D-galactose. These effects can be exploited to purify hexokinase from crude yeast extracts. 6. The specific active-site-directed binding of triazine dyes to yeast hexokinase is interpreted in terms of the crystallographic structure of the hexokinase monomer.     

Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.     

Hexose-specificity of hexokinase and ADP-dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoa

Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 +/- 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 +/- 0.1 mIU/mg protein to 0.60 +/- 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15-0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface.     

Identification of a novel casein kinase activity in HeLa cell nuclei

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.     

Emergence of pyridoxal phosphorylation through a promiscuous ancestor during the evolution of hydroxymethyl pyrimidine kinases

In the family of ATP-dependent vitamin kinases, several bifunctional enzymes that phosphorylate hydroxymethyl pyrimidine (HMP) and pyridoxal (PL) have been described besides enzymes specific towards HMP. To determine how bifunctionality emerged, we reconstructed the sequence of three ancestors of HMP kinases, experimentally resurrected, and assayed the enzymatic activity of their last common ancestor. The latter has ∼8-fold higher specificity for HMP due to a glutamine residue (Gln44) that is a key determinant of the specificity towards HMP, although it is capable of phosphorylating both substrates. These results show how a specific enzyme with catalytic promiscuity gave rise to current bifunctional enzymes.