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)

The truncated gene of hexokinase, mini-hexokinase, starting with methionine 455 and ending at the C terminus was expressed in Escherichia coli. Mini-hexokinase lost its ability to ameliorate inhibition of glucose-6-P-inhibited mini-hexokinase in the presence of phosphate (P(i)). We suggest that the P(i) site either resides in the N-terminal half of hexokinase I or requires the N-terminal portion of the enzyme. Site-directed mutagenesis was performed to obtain two mutants of mini-hexokinase: C606S and C628S. Both are thought to be associated with the active site of hexokinase I. These mutants exhibited a 3-fold increase in Km for glucose but no change in either the Km for ATP or the kcat. The circular dichroism (CD) spectra showed no differences among the wild-type or mutant enzymes. These results suggest that Cys606 and Cys628 are not involved in glucose binding directly. The putative ATP-binding site of full-length human brain hexokinase may involve Arg539 and Gly679, and these residues were mutated to Ile. For the mutant R539I, the kcat value decreased 114-fold relative to wild-type hexokinase, whereas the Km values for ATP and glucose changed only slightly. No change was observed in the Ki value for 1,5-anhydroglucitol 6-phosphate. CD spectra showed only a slight change in secondary structure. For the mutant G679I, overexpressed hexokinase is insoluble. We suggest that Arg539 is important for catalysis because it stabilizes the transition state product ADP-hexokinase. Gly679 is probably important for proper folding of the protein.
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PMID:Active site residues of human brain hexokinase as studied by site-specific mutagenesis. 773 85

A Xenopus oocyte expression system was used to examine how glucose transporters (GLUT 2 and GLUT 3) and glucokinase (GK) activity affect glucose utilization. Uninjected oocytes and low rates of both glucose transport and phosphorylation; expression of GLUT 2 or GLUT 3 increased glucose phosphorylation approximately 20-fold by a low Km, endogenous hexokinase at glucose concentrations < or = 1 mM, but not at higher glucose concentrations. Coexpression of functional GK isoforms with GLUT 2 or 3 increased glucose utilization approximately an additional two- to threefold primarily at the physiologic glucose concentrations of 5-20 mM. The Km for glucose of both the hepatic and beta cell isoforms of GK, determined in situ, was approximately 5-10 mM when coexpressed with either GLUT 2 or GLUT 3. The increase in glucose utilization by coexpression of GLUT 3 and GK was dependent upon glucose phosphorylation since two missense GK mutations linked with maturity-onset diabetes, 182: Val-->Met and 228:Thr-->Met, did not increase glucose utilization despite accumulation of both a similar amount of immunoreactive GK protein and glucose inside the cell. Coexpression of a mutant GK and a normal GK isoform did not interfere with the function of the normal GK enzyme. Since the coexpression of GK and a glucose transporter in oocytes resembles conditions in the hepatocyte and pancreatic beta cell, these results indicate that increases in glucose utilization at glucose concentrations > 1 mM depend upon both a functional glucose transporter and GK.
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PMID:Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization. 792 12

This study followed changes in the capacities of uptake and phosphorylation of glucose in response to contractile activity in low-frequency stimulated (10Hz, 24 h/d) rat fast-twitch muscle. We investigated the intracellular distribution of GLUT-4, the major glucose transporter isoform in muscle, changes in the amounts of its specific mRNA and total cellular protein, as well as changes in its relative synthesis rate. These analyses were complemented by measurements of total hexokinase activity and hexokinase II (HKII) expression at the levels of mRNA content and protein synthesis. Changes in protein synthesis were determined by in vivo labeling with [35S]methionine. Translocation of GLUT-4 into the sarcolemma was an immediate response to contractile activity, whereas changes in its total amount were observed only with ongoing stimulation (5 d and longer). A twofold increase in GLUT-4 content after 5 d and longer stimulation periods was preceded by elevations of its mRNA and by enhanced [35S]methionine incorporation. Conversely, increases in HKII expression with a rise in total hexokinase activity occurred soon after the onset of stimulation (30-fold elevations of HKII mRNA after 12 h and 20-fold increases in [35S]methionine incorporation after 24 h). With ongoing stimulation, HKII mRNA and synthesis returned to lower levels (fivefold elevations). Nevertheless, hexokinase activity continued to rise, stabilizing at fivefold-elevated levels after 3 d. These observation suggested that posttranscriptional mechanisms contributed to the upregulation of HKII, e.g. stabilization by elevated intracellular glucose and mitochondrial binding of the enzyme. This suggestion was supported by experiments with cessation after 24 h where hexokinase activity continued to increase, although the mRNA content and, especially, the [35S]methionine incorporation decayed steeply. The increase in HKII prior to GLUT-4 suggests that phosphorylation may be rate limiting in glucose utilization of glycolytic fibers under conditions of sustained contractile activity. Taken together, the changes in distribution and content of GLUT-4, as well as in HKII represent early metabolic adaptations. In addition, they are related to the overall process of stimulation-induced fiber type transformation.
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PMID:Low-frequency stimulation of rat fast-twitch muscle enhances the expression of hexokinase II and both the translocation and expression of glucose transporter 4 (GLUT-4). 830 97

It was previously reported that inhibition of carnitine synthesis by 3-(2,2,2-trimethyl-hydrazinium) propionate (MET-88) restores left ventricular (LV) systolic and diastolic function in rats with myocardial infarction (MI). Preservation of the calcium uptake function of sarcoplasmic reticulum Ca2+-ATPase (SERCA2) is one of the possible mechanisms by which MET-88 alleviates hemodynamic dysfunction. To test this hypothesis, the effects of MET-88 on protein content of SERCA2 were evaluated using the same rat model of heart failure. Myocardial protein content of hexokinase, which is one of the key enzymes of glucose utilization, was also measured. Either MET-88 (MET-88 group) or a placebo (MI group) was administered for 20 days to rats with MI induced by coronary artery ligation. The control group underwent sham surgery (no ligation) and received placebo. In LV myocardial homogenates, the myocardial SERCA2 protein content was 32% lower (p<0.05) in the MI group than in the control group. However, in the MET-88 group myocardial SERCA2 content was the same as in the control group. Hexokinase I protein content was 29 % lower (p<0.05) in the MI group compared with the control. In contrast, hexokinase II protein content did not differ significantly among the three groups. Consequently, inhibition of carnitine synthesis ameliorates depression of SERCA2 and hexokinase I protein content which may reduce tissue damage caused by MI.
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PMID:Inhibition of carnitine synthesis modulates protein contents of the cardiac sarcoplasmic reticulum Ca2+-ATPase and hexokinase type I in rat hearts with myocardial infarction. 1109 60

To assess what properties of glucose metabolism are most closely related to expression of the neural phenotype, some parameters of glucose metabolism in PC12 cells before (tumor-type) and after differentiation (neuron-type) were investigated. Neuron-type cells exhibited a 2.7-fold higher level of [3H]DG retention than tumor-type cells, accompanied by a higher glucose transport rate and higher levels of hexokinase activity. [14C]CO2 production from [U-14C]glucose in neuron-type was also more than four-times greater than that in tumor-type cells. The levels of [14C]carbon in macromolecules from [14C]glucose in neuron-type cells were also much higher (10.6-fold) than those in tumor-type cells, and the levels of incorporation of [14C]carbon were almost as high as those of [14C]CO2. From the metabolite analysis, amino acids appeared to be the major compounds converted from glucose. On the other hand, the uptakes of [35S]methionine-[35S]cysteine and [3H]uridine in neuron-type cells were lower than those in tumor-type cells. Following depolarization with 50 mM potassium, [14C]CO2 production increased, but the retention of [14C]carbon was not changed in neuron-type cells. The largest change accompanied by acquisition of the neural phenotype was carbon incorporation into the macromolecules derived from glucose. This property may be important for the expression of the neural phenotype as well as the higher levels of both glucose uptake and oxygen consumption.
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PMID:Dynamic changes in glucose metabolism accompanying the expression of the neural phenotype after differentiation in PC12 cells. 1124 18

The cAMP-dependent protein kinase anchoring protein, d-AKAP1, has two N-terminal splice variants. The shorter forms (N0, d-AKAP1a, and -1c) target to mitochondria, and the longer forms (N1, d-AKAP1b, and -1d) with 33 additional residues N-terminal to N0 target to the endoplasmic reticulum (ER) (Huang, L. J., Wang, L., Ma, Y., Durick, K., Perkins, G., Deerinck, T. J., Ellisman, M. H., and Taylor, S. S. (1999) J. Cell Biol. 145, 951-959). In d-AKAP1a, translation may initiate from both Met-34 or Met-49 producing two molecules both targeted to mitochondria. The shorter molecule contains the 15-residue targeting motif, homologous to the N-terminal mitochondrial targeting motif of hexokinase I. Extensive mutagenesis showed that one hydrophobic surface of the 15-residue hexokinase-homologous segment contained the key elements for mitochondrial targeting. The same 15 residues are also part of the ER-targeting signal, but for ER targeting multiple hydrophobic residues are required that encompass both surfaces of the helix. The different involvement of the same helical motif for targeting to the two organelles appears to reflect different modes of interaction with the two organelles. This is the first example of a bifunctional helical element that is required for both ER and mitochondrion targeting.
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PMID:A 15-residue bifunctional element in D-AKAP1 is required for both endoplasmic reticulum and mitochondrial targeting. 1199 83

One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.
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PMID:Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria. 1616 83

1. Ethionine-treated mice showed a marked depletion in liver glycogen, a decrease of glycogen-synthetase activity, an increase in activity of glucose 6-phosphate dehydrogenase and the solubilization of phosphorylase. 2. The administration of cortisol or glucose did not alleviate these changes but the effect of ethionine was completely prevented in animals given methionine as well as ethionine. 3. The activities of the following enzymes were unchanged: hexokinase, glucokinase, glucose 6-phosphatase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase and pyruvate kinase.
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PMID:Depletion of glycogen synthetase and increase of glucose 6-phosphate dehydrogenase in livers of ethionine-treated mice. 1674 21

Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V(max) with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.
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PMID:Kinetic and proteomic analyses of S-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism. 1705 22

The yeast protein kinases Sat4/Hal4 and Hal5 are required for the plasma membrane stability of the K(+) transporter Trk1 and some amino acid and glucose permeases. The transcriptomic analysis presented here indicates alterations in the general control of the metabolism of both nitrogen and carbon. Accordingly, we observed reduced uptake of methionine and leucine in the hal4 hal5 mutant. This decrease correlates with activation of the Gcn2-Gcn4 pathway, as measured by expression of the lacZ gene under the control of the GCN4 promoter. However, with the exception of methionine biosynthetic genes, few amino acid biosynthetic genes are induced in the hal4 hal5 mutant, whereas several genes involved in amino acid catabolism are repressed. Concerning glucose metabolism, we found that this mutant exhibits derepression of respiratory genes in the presence of glucose, leading to an increased activity of mitochondrial enzymes, as measured by succinate dehydrogenase (SDH) activity. In addition, the reduced glucose consumption in the hal4 hal5 mutant correlates with a more acidic intracellular pH and with low activity of the plasma membrane H(+)-ATPase. As a compensatory mechanism for the low glycolytic rate, the hal4 hal5 mutant overexpresses the HXT4 high-affinity glucose transporter and the hexokinase genes. These results indicate that the hal4 hal5 mutant presents defects in the general control of nitrogen and carbon metabolism, which correlate with reduced transport of amino acids and glucose, respectively. A more acidic intracellular pH may contribute to some defects of this mutant.
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PMID:Hal4 and Hal5 protein kinases are required for general control of carbon and nitrogen uptake and metabolism. 2095 80


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