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)

Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration. The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E. coli enzyme. Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction. The most logical explanation of the results with the E. coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction. The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E. coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release.
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PMID:Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase. 0 91

It is demonstrated that N-bromoacetyl-D-galactosamine acts as a substrate-like reagent for yeast hexokinases A and B, producing affinity labeling. At the order of 10(-3) M reagent concentrations, rapid inactivation of the enzyme is produced: the kinetics are consistent with dependence upon a reversible inhibitor-enzyme initial complex, with a dissociation constant of 3.8 x 10(-3) M for hexokinase B at 35 degrees, pH 8.5. The glucose analog is 30-fold less effective, presumably due to self-protection. The inactivating reaction is an order of magnitude faster than that with bromoacetate. All the alkylation of hexokinase B was shown to occur at two thiol groups per subunit, associated stoichiometrically with inactivation. Unlike the reaction there of simple alkylators, two nonessential thiols per subunit are left unattacked when this inactivation reaction is complete. Protection against the affinity alkylation is exerted by the substrates glucose, mannose, fructose, glucose 6-phosphate, fructose 6-phosphate, ATP-Mg, and ADP-Mg, in proportion to their affinities for the active center. Free ATP also protects. Mg2+ alone has no influence, and Mn2+ gives a slight acceleration, when correction is made for a slow inactivation that occurs when the enzyme is incubated at 35 degrees with Mn2+ alone. Galactose, virtually a nonsubstrate, has no influence on the affinity alkylation, but N-acetylgalactosamine, a nonsubstrate and a weak inhibitor of the enzymic reaction, has an accelerating effect. An interpretation is made in terms of binding to a site that influences the active center. This affinity label should provide a means of isolating a peptide containing active-center-related groups.
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PMID:Essential thiols of yeast hexokinase: alkylation by a substrate-like reagent. 109 53

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously [Jarori, G. K., Kasturi, S. R. & Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258-268]. The paramagnetic effect of enzyme-bound Mn(II) on the spin-lattice relaxation rates (T-1(1] of ligand nuclei (1H and 31P) in E.Mn(II).Glc and E.Mn(II).Glc6P complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-H alpha, C1-H beta and C2-H beta of glucose in the E.Mn(II).Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5-30 degrees C at 500 MHz. The ligand nuclear relaxation rates in E.Mn(II).Glc are field-dependent and the Arrhenius plot yields an activation energy (delta E) of 16.7-20.9 kJ/mol. Similar measurements have also been carried out on C1-H alpha, C1-H beta and C6-31P at 270 MHz (1H) and 202.5 MHz (31P) for the E.Mn(II).Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5-30 degrees C, which yielded delta E = 9.2 kJ/mol. The electron-nuclear dipolar correlation time (tau c), determined from the field-dependent measurements of proton relaxation rates in the E.Mn(II).Glc complex, is 0.22-1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are approximately 1.1 nm and approximately 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare, U. W. (1988) J. Biol. Chem. 263, 15492-15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P.Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of approximately 0.5-0.6 nm from the cation.
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PMID:Mapping of glucose and glucose-6-phosphate binding sites on bovine brain hexokinase. A 1H- and 31P-NMR investigation. 231 6

Rat brain cytosolic and mitochondrial hexokinase activities were undetectable without added divalent cations. Mg2+ activated cytosolic (K0.5 of Mg2+ = 343 +/- 13 microM) and mitochondrial (K0.5 of Mg2+ = 183 +/- 8 microM) hexokinase in a concentration-related manner. The corresponding values for Mn2+ were 702 +/- 99 and 413 +/- 21 microM respectively. Ca2+, however, activated both forms of hexokinase poorly. In the presence of Mg2+, both Mn2+ and Cu2+ were more potent inhibitors of cytosolic hexokinase than mitochondrial hexokinase, whereas the inhibition of Cd2+ and Ca2+ did not show such selectivity. These results demonstrate that brain mitochondrial and cytosolic hexokinases differ significantly in their responses to divalent cations.
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PMID:Differences in the responses of brain cytosolic and mitochondrial hexokinases to three essential divalent metal ions. 286 Oct 10

The effects of monovalent (Li+, Cs+) divalent (Cu2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Hg2+, Pb2+, Mn2+, Fe2+, Co2+, Ni2+) and trivalent (Cr3+, Fe3+, Al3+) metals ions on hexokinase activity in rat brain cytosol were compared at 500 microM. The rank order of their potency as inhibitors of brain hexokinase was: Cr3+ (IC50 = 1.3 microM) greater than Hg2+ = Al3+ greater than Cu2+ greater than Pb2+ (IC50 = 80 microM) greater than Fe3+ (IC50 = 250 microM) greater than Cd2+ (IC50 = 540 microM) greater than Zn2+ (IC50 = 560 microM). However, at 500 microM Co2+ slightly stimulated brain hexokinase whereas the other metal ions were without effect. That inhibition of brain glucose metabolism may be an important mechanism in the neurotoxicity of metals is suggested.
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PMID:Differential effects of monovalent, divalent and trivalent metal ions on rat brain hexokinase. 286 Oct 11

Manganese causes a significant rise in hepatic glucokinase and hexokinase in 16-day-old suckling rats, and has an insulinomimetic effect in producing a precocious emergence of glucokinase (EC 2.7.1.2) and a rise in the low Km, hexokinases (EC 2.7.1.1) activities. These enzyme changes occur within 6 hr of manganese administration and there are accompanying increases in plasma insulin and hepatic cyclic GMP. That the effect of manganese is at a site other than, or in addition to, insulin secretion is suggested by the significant increases in glucokinase and hexokinase in 16-day-old streptozotocin-diabetic rats; in this group there is also an increase in hepatic cGMP similar in time scale to that of the normal-manganese-treated group. The effects of manganese and insulin were not additive. It is proposed that one site of action of manganese may be at the level of cyclic GMP systems. The results are also discussed in relation to the known action of manganese at the level of the protein phosphatases.
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PMID:The insulin-mimetic action of Mn2+: involvement of cyclic nucleotides and insulin in the regulation of hepatic hexokinase and glucokinase. 299 12

Autophosphorylation of hexokinase PII was studied using an enzyme purified from Saccharomyces cerevisiae. Incubation of this enzyme preparation with [gamma-32P]ATP and Mn2+ or Mg2+ gave a phosphoprotein of molecular mass 58,000 which corresponded to hexokinase PII. D-Xylose stimulated autophosphorylation of hexokinase PII. Dilution of hexokinase PII over a 10-fold concentration range did not change the specific activity of hexokinase PII autophosphorylation suggesting that it may occur by an intramolecular mechanism.
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PMID:Autophosphorylation of yeast hexokinase PII. 307 85

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.
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PMID:Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes. 312 60

The mechanism of retraction of the longitudinal flagellum of Ceratium tripos was studied by making extracted models of the flagellum. Non-detergent models extracted in low ionic strength medium containing 1 M-glucose, 10 mM-EDTA, and 50 mM-Tris X HCl buffer (pH 8.0), retracted when Ca2+, Mg2+, Ba2+, Sr2+, Mn2+ or Cd2+ was applied locally with a glass capillary. A demembranated model of the flagellum was made with an extraction medium containing 0.8-1.0 M-glucose, 20 mM-Tris-acetate (pH 7.8), 2 mM-EGTA, 5-7 mM-MgSO4, 0.1 M-potassium glutamate and 0.1% Triton X-100. The model required a concentration of Mg2+ of a few mmol/l for successful reactivation of both retraction and undulation, and about 0.1 M-potassium glutamate (or sodium glutamate) for reactivation of undulation. Neither type of motion of the models could be reactivated above 35 degrees C. Ca2+ induced the retraction at pCa 5.5 or less. In addition to Ca2+, Mn2+, Ba2+, Sr2+ and Cd2+ also induced retraction but Mg2+, La3+ or Tb3+ did not. Although ATP was required for undulation, it was not required for retraction. Co-incubation with hexokinase to remove contaminating ATP did not suppress the retraction. The potent ATPase inhibitor, orthovanadate, inhibited undulation at 10 micron but did not inhibit retraction even at 2 mM. SH blockers, N-ethylmaleimide and dithio-bis-nitrobenzoic acid strongly suppressed undulation but had no effect on retraction. Calmodulin inhibitors, trifluoperazine and chlorpromazine, also had no effect on retraction. These data indicate that undulation is generated by a 9 + 2 microtubular axoneme using energy released by hydrolysis of ATP and that retraction can be induced by Ca2+ without a requirement for ATP.
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PMID:Extraction model of the longitudinal flagellum of Ceratium tripos (Dinoflagellida): reactivation of flagellar retraction. 385 92

Our previous studies have shown that one manganous ion binds tightly to bovine hexokinase, with a Kd = 25 +/- 4 microM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (epsilon b) is 3.5 at 9 MHz and 23 degrees C [Jarori, G.K. Kasturi, S.R., and Kenkare, U.W. (1981) Arch. Biochem. Biophys. 211, 258-268]. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E X Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme X nucleotide X Mn. Addition of glucose 6-phosphate to enzyme X ATP X Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E X ADP X Mn X Glc-6-P appears to be formed when both the products are present. Beta, gamma-Bidentate Cr(III)ATP has been used to elucidate the role of direct binding of Mn(II) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(III)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron-spin resonance studies on the binding of Mn(II) to the enzyme in the presence of Cr(III)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis. Experiments carried out with hexokinase spin-labeled with 3-(2-iodo-acetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site.
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PMID:Magnetic resonance studies on the interaction of metal-ion and nucleotide ligands with brain hexokinase. 609 Jan 39


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