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)

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.
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PMID:Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria. 1524

Intracellular glucose in Escherichia coli cells imported by phosphoenolpyruvate-dependent phosphotransferase system-independent uptake is phosphorylated by glucokinase by using ATP to yield glucose-6-phosphate. Glucokinases (EC 2.7.1.2) are functionally distinct from hexokinases (EC 2.7.1.1) with respect to their narrow specificity for glucose as a substrate. While structural information is available for ADP-dependent glucokinases from Archaea, no structural information exists for the large sequence family of eubacterial ATP-dependent glucokinases. Here we report the first structure determination of a microbial ATP-dependent glucokinase, that from E. coli O157:H7. The crystal structure of E. coli glucokinase has been determined to a 2.3-A resolution (apo form) and refined to final Rwork/Rfree factors of 0.200/0.271 and to 2.2-A resolution (glucose complex) with final Rwork/Rfree factors of 0.193/0.265. E. coli GlK is a homodimer of 321 amino acid residues. Each monomer folds into two domains, a small alpha/beta domain (residues 2 to 110 and 301 to 321) and a larger alpha+beta domain (residues 111 to 300). The active site is situated in a deep cleft between the two domains. E. coli GlK is structurally similar to Saccharomyces cerevisiae hexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs. Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1. Glucose binding results in a closure of the small domains, with a maximal Calpha shift of approximately 10 A. A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the O6 hydroxyl of glucose, followed by nucleophilic attack at the gamma-phosphoryl group of ATP, yielding glucose-6-phosphate as the product.
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PMID:Crystal structures of Escherichia coli ATP-dependent glucokinase and its complex with glucose. 1546 45

Several studies have shown impairment of neutrophil function, a disorder that contributes to the high incidence of infections in diabetes. Since glucose and glutamine play a key role in neutrophil function, we investigated their metabolism in neutrophils obtained from the peritoneal cavity of streptozotocin-induced diabetic rats. The activities of hexokinase, glucose-6-phosphate dehydrogenase (G6PDH), phosphofructokinase (PFK), citrate synthase, phosphate-dependent glutaminase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were assayed. Glucose, glutamine, lactate, glutamate and aspartate, and the decarboxylation of [U-14C], [1-14C] and [6-14C]glucose; [U-14C]palmitic acid; and [U-14C]glutamine were measured in 1-h incubated neutrophils. Phagocytosis capacity and hydrogen peroxide (H2O2) production were also determined. All measurements were carried out in neutrophils from control, diabetic and insulin-treated (2-4 IU/day) diabetic rats. Phagocytosis and phorbol myristate acetate (PMA)-stimulated H2O2 production were decreased in neutrophils from diabetic rats. The activities of G6PDH and glutaminase were decreased, whereas that of PFK was raised by the diabetic state. The activities of the remaining enzymes were not changed. Diabetes decreased the decarboxylation of [1-14C]glucose and [U-14C]glutamine; however, [6-14C]glucose and [U-14C]palmitic acid decarboxylation was increased. These observations indicate that changes in metabolism may play an important role in the impaired neutrophil function observed in diabetes. The treatment with insulin abolished the changes induced by the diabetic state even with no marked change in glycemia. Therefore, insulin may have a direct effect on neutrophil metabolism and function.
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PMID:Diabetes causes marked changes in function and metabolism of rat neutrophils. 1646 55

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO(3) or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.
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PMID:Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes. 1666 66

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.
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PMID:Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues. 1700 45

HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, (31)P NMR, and (1)H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca(2+) in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.
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PMID:The interference of HEPES buffer during amperometric detection of ATP in clinical applications. 1836 90

Mitochondrial hexokinase (mt-HK) and creatine kinase (mt-CK) activities have been recently proposed to reduce the rate of mitochondrial ROS generation through an ADP re-cycling mechanism. Here, we determined the role of mt-HK and mt-CK activities in regulate mitochondrial ROS generation in rat brain, kidney, heart and liver, relating them to the levels of classical antioxidant enzymes. The activities of both kinases were significantly higher in the brain than in other tissues, whereas the activities of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were higher in both liver and kidney mitochondria. In contrast, manganese superoxide dismutase (Mn-SOD) activity was not significantly different among these tissues. Activation of mitochondrial kinases by addition of their substrates increased the ADP re-cycling and thus the respiration by enhancing the oxidative phosphorylation. Succinate induced hydrogen peroxide (H(2)O(2)) generation was higher in brain than in kidney and heart mitochondria, and the lowest in liver mitochondria. Mitochondrial membrane potential (DeltaPsi(m)) and H(2)O(2) production, decreased with additions of 2-DOG or Cr to respiring brain and kidney mitochondria but not to liver. The inhibition of H(2)O(2) production by 2-DOG and Cr correspond to almost 100% in rat brain and about 70% in kidney mitochondria. Together our data suggest that mitochondrial kinases activities are potent preventive antioxidant mechanism in mitochondria with low peroxidase activities, complementing the classical antioxidant enzymes against oxidative stress.
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PMID:Reactive oxygen species generation is modulated by mitochondrial kinases: correlation with mitochondrial antioxidant peroxidases in rat tissues. 1863 44

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (KMGlc=140 microM versus KMFrc=1,375 microM). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H2O2) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H2O2 release. The blockage of H2O2 release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F0F1ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H2O2 release. Inhibition in H2O2 release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H2O2 release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H2O2 release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.
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PMID:Reactive oxygen species production by potato tuber mitochondria is modulated by mitochondrially bound hexokinase activity. 1910 13

Jasmonates act as signal transduction intermediates when plants are subjected to environmental stresses such as UV radiation, osmotic shock and heat. In the past few years several groups have reported that jasmonates exhibit anti-cancer activity in vitro and in vivo and induce growth inhibition in cancer cells, while leaving the non-transformed cells intact. Recently, jasmonates were also discovered to have cytotoxic effects towards metastatic melanoma both in vitro and in vivo. Three mechanisms of action have been proposed to explain this anti-cancer activity. The bio-energetic mechanism - jasmonates induce severe ATP depletion in cancer cells via mitochondrial perturbation. Furthermore, methyl jasmonate (MJ) has the ability to detach hexokinase from the mitochondria. Second, jasmonates induce re-differentiation in human myeloid leukemia cells via mitogen-activated protein kinase (MAPK) activity and were found to act similar to the cytokinin isopentenyladenine (IPA). Third, jasmonates induce apoptosis in lung carcinoma cells via the generation of hydrogen peroxide, and pro-apoptotic proteins of the Bcl-2 family. Combination of MJ with the glycolysis inhibitor 2-deoxy-d-glucose (2DG) and with four conventional chemotherapeutic drugs resulted in super-additive cytotoxic effects on several types of cancer cells. Finally, jasmonates have the ability to induce death in spite of drug-resistance conferred by either p53 mutation or P-glycoprotein (P-gp) over-expression. In summary, the jasmonates are anti-cancer agents that exhibit selective cytotoxicity towards cancer cells, and thus present hope for the development of cancer therapeutics.
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PMID:Methyl jasmonate: a plant stress hormone as an anti-cancer drug. 1966 Jul 69

Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.
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PMID:Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana. 2144 6


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