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)

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20-30% of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 X 10(6). Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 X 10(6), which contained a proportion of all the glycolytic enzyme studied; the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1 600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6-phosphate was reduced by the presence of unlabelled 3-phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope investigation of both systems showed large numbers of regular 30 nm diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.
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PMID:The tentative identification in Escherichia coli of a multienzyme complex with glycolytic activity. 13

Yeast hexokinase is rapidly inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosyl ethyl ester. Sugar substrates afford a partial protection, which is increased by the addition of ADP. Inactivation of the enzyme takes place concomitantly with the incorporation of 1 mol of nitrotyrosine per mol of 50 000-dalton subunit. Exhaustive proteolytic digestion of the modified protein and isolation of the nitrotyrosyl peptide by affinity chromatography, followed by electrophoresis, lead to the identification of the modified residue as a glutamyl residue. This modification of hexokinase occurs without gross conformational changes. The enzyme still binds its substrates, though binding of the nucleotides is perturbed. While the substrates afford a partial protection, they increase the incorporation of nitrotyrosine ethyl ester into the enzyme. This may be attributed to local conformational changes which their binding induces. It is concluded that a glutamyl residue is essential for yeast hexokinase activity and its catalytic function is discussed.
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PMID:Evidence for an essential glutamyl residue in yeast hexokinase. 33 45

Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a 'half-of-the-sites' mechanism. 284 99

Triethyltin bromide was found to demonstrate temperature-dependent inactivation of yeast hexokinase B. At temperatures of 20 degrees C or lower, little or no inactivation of the enzyme was detected after 2 h of reaction with 50-300 microM concentrations of the reagent. However, incubation at 25 degrees C or higher resulted in an increased rate and extent of loss of the enzyme activity with increasing incubation temperatures. The Arrhenius plot for the inactivation process showed a sharp break at approximately 30 degrees C, with a heat of activation (delta H*) above this temperature of 55.2 kcal, indicating that a triethyltin-induced conformational change occurred at the elevated temperatures. Sugar substrates provided protection against the inactivating effect by reducing the binding of triethyltin to the enzyme. In the absence of glucose, two sites of different affinity for triethyltin exist in the hexokinase monomer. Binding of triethyltin to the enzyme shifted its monomer-dimer equilibrium toward the monomeric form in an early stage of the interaction. Inactivation of the enzyme was associated with a slower subsequent event. Comparative effects of various organotin compounds on the activity of the enzyme indicated that inhibitory potency was associated with increasing hydrophobicity of the alkyl groups attached to the tin.
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PMID:Inactivation of yeast hexokinase B by triethyltin bromide. 622 11

Sugar repression of photosynthetic genes is likely a central control mechanism mediating energy homeostasis in a wide range of algae and higher plants. It overrides light activation and is coupled to developmental and environmental regulations. How sugar signals are sensed and transduced to the nucleus remains unclear. To elucidate sugar-sensing mechanisms, we monitored the effects of a variety of sugars, glucose analogs, and metabolic intermediates on photosynthetic fusion genes in a sensitive and versatile maize protoplast transient expression system. The results show that sugars that are the substrates of hexokinase (HK) cause repression at a low concentration (1 to 10 mM), indicating a low degree of specificity and the irrelevance of osmotic change. Studies with various glucose analogs suggest that glucose transport across the plasma membrane is necessary but not sufficient to trigger repression, whereas subsequent phosphorylation by HK may be required. The effectiveness of 2-deoxyglucose, a nonmetabolizable glucose analog, and the ineffectiveness of various metabolic intermediates in eliciting repression eliminate the involvement of glycolysis and other metabolic pathways. Replenishing intracellular phosphate and ATP diminished by hexoses does not overcome repression. Because mannoheptulose, a specific HK inhibitor, blocks the severe repression triggered by 2-deoxyglucose and yet the phosphorylated products per se do not act as repression signals, we propose that HK may have dual functions and may act as a key sensor and signal transmitter of sugar repression in higher plants.
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PMID:Sugar sensing in higher plants. 782 98

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promoter was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.
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PMID:Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). 789 14

Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by 'Kitz-Wilson'-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inactivation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations. 819 58

Sucrose is the main transported form of assimilates, but, significantly, it also regulates a variety of processes such as photosynthesis and carbon or nitrogen storage. The effects of high sucrose levels are mediated directly by modulation of gene expression. The regulation of storage protein accumulation, here patatin from potato tubers, was used as a model system to study sucrose mediated signal transduction. The transcriptional regulation of patatin genes in conserved in transgenic Arabidopsis, as shown by the analysis of expression of two classes of patatin promoters fused to uidA. Two distinctly different patterns of gene expression were observed. In roots, class I promoter expression is strongly dependent on the exogenous supply of sugars. 3-O-methylglucose induction indicates that the sensor is located upstream of hexokinase. In contrast, the class II promoter is constitutively active in root tips and hydatodes. The progeny of a homozygous class I line was mutagenized with ethyl methane sulphonate and screened for signal transduction mutants using a non-destructive screening system for GUS activity. Four mutants showing reduced sucrose responses (rsr) and two mutants with modified expression patterns (mep) regarding the root tip were identified. In backcross analyses, it was shown that rsr1-1 carries a recessive trans mutation whereas rsr4-1 seems to be a semi-dominant trans mutation in sugar-mediated gene regulation.
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PMID:Identification of mutants in metabolically regulated gene expression. 902 2

The proton-sucrose symporter mediates the key transport step in the resource distribution system that allows many plants to function as multicellular organisms. In the results reported here, we identify sucrose as a signaling molecule in a previously undescribed signal-transduction pathway that regulates the symporter. Sucrose symporter activity declined in plasma membrane vesicles isolated from leaves fed exogenous sucrose via the xylem transpiration stream. Symporter activity dropped to 35-50% of water controls when the leaves were fed 100 mM sucrose and to 20-25% of controls with 250 mM sucrose. In contrast, alanine symporter and glucose transporter activities did not change in response to sucrose treatments. Decreased sucrose symporter activity was detectable after 8 h and reached a maximum by 24 h. Kinetic analysis of transport activity showed a decrease in Vmax. RNA gel blot analysis revealed a decrease in symporter message levels, suggesting a drop in transcriptional activity or a decrease in mRNA stability. Control experiments showed that these responses were not the result of changing osmotic conditions. Equal molar concentrations of hexoses did not elicit the response, and mannoheptulose, a hexokinase inhibitor, did not block the sucrose effect. These data are consistent with a sucrose-specific response pathway that is not mediated by hexokinase as the sugar sensor. Sucrose-dependent changes in the sucrose symporter were reversible, suggesting this sucrose-sensing pathway can modulate transport activity as a function of changing sucrose concentrations in the leaf. These results demonstrate the existence of a signaling pathway that can control assimilate partitioning at the level of phloem translocation.
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PMID:Sucrose is a signal molecule in assimilate partitioning. 953 16

Sugar-mediated regulation of gene expression is a mechanism controlling the expression of many different plant genes. In this review, a compilation of the genes encoding photosynthetic proteins, subject to this mode of regulation, is presented. Several groups have devised different screening strategies to obtain Arabidopsis mutants in sugar sensing and signalling. An overview of these strategies has been included. Sugar-mediated regulation of gene expression is thought to require the hexokinase (HXK) protein. It has previously been shown that one such sugar, mannose, is capable of blocking germination in Arabidopsis. This inhibition is also mediated by HXK and occurs in the low millimolar concentration range. Here, the use of germination on mannose as an effective screening strategy for putative sugar sensing and signalling mutants is reported. T-DNA- and EMS-mutagenized collections were used to isolate 31 mannose-insensitive germination (mig) mutants. With the use of these mutants, a comparison between this screen and other existing sugar-sensing screens is presented.
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PMID:Photosynthesis, sugars and the regulation of gene expression. 1093 49


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