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 activities of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, G6PD), 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate: NADP oxidoreductase, 6PGD), hexokinase (ATP:D-hexose 6-phosphotransferase, HK), lactic dehydrogeanse (L-lactate: NAD oxidoreductase, LDH) and aspirate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, Asp.T) were determined in red blood cells of 11 healthy individuals. The determinations were carried out on samples drawn every 4 h over a 24 h period. The activities of G6PD, 6PGD, LDH and Asp.T exhibited a semi-circadian rhythm, namely, two peaks of activity during 24 h while HK activity demonstrated a true circadian rhythm. In addition a polymorphism of the G6PD and LDH activity patterns was observed. The implications of a biological clock in enucleated cells are discussed.
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PMID:The diurnal rhythm of enzymes in human red cells. 94 47

An examination of the binding sites of four carbohydrate binding proteins (Escherichia coli lactose repressor, E. coli arabinose-binding protein, yeast hexokinase A and Concanavalin A) revealed certain similarities of amino acid sequences and residues forming hydrogen bonds and hydrophobic interactions with the bound carbohydrate. These were: (i) Asx-Asx, hydrogen bonding to the pyranose ring oxygen and anomeric-OH group; (ii) Arg-X-X-X-(Ser/Thr), or the reverse sequence, with the Arg hydrogen bonding to the pyranose ring oxygen; (iii) Lys-(Ser/Thr)-X-X-Asp, or the reverse sequence and with interchange of the Lys-(Ser/Thr) positions, with hydrogen bonding of either or both the Lys and Asp residues to the -OH groups at carbons 2, 3, 4 or 6; (iv) a diaromatic sequence with possible hydrophobic interactions to the faces of the pyranose ring structure. An algorithm was devised to search the amino acid sequences of a large number of proteins, those known to bind carbohydrates as well as those without known carbohydrate-binding activities, for the four amino acid sequence criteria. The algorithm incorporated a weighted distance value (WDV) to assess the approximate distance between any two criteria, with the WDV being based on the predicted secondary structure of the protein amino acid sequence. When the algorithm using criteria 1 and 2 plus the WDV was applied to the sequences of 125 proteins, the method indicated the presence of the potential carbohydrate-binding site motif for 42% of proteins with known carbohydrate binding, only 8% of proteins were predicted as false positives, and the accuracy of the method was calculated to be 61.6%.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A method for identifying a proposed carbohydrate-binding motif of proteins. 182 33

Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.
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PMID:Expression and site-directed mutagenesis of hepatic glucokinase. 185 32

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type. The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.
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PMID:Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase. 200 85

Yeast hexokinase B (ATP:-hexose 6-phosphotransferase, EC 2.7.1.1) was crystallized in the presence of D-xylose and ADP, and its structure was determined at 7 A resolution. The enzyme is in the 'open' conformation which is characteristic of the enzyme crystallized in the absence of glucose, rather than in the 'closed' conformation that is observed with the glucose complex. That is, the binding of xylose into the large cleft that separates the molecule into two lobes does not cause the cleft to close. We conclude, then, that the glucose 6-hydroxymethyl group (which binds to an aspartic acid and a serine) is essential for the hexose-induced conformational change.
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PMID:The 6-hydroxymethyl group of a hexose is essential for the substrate-induced closure of the cleft in hexokinase. 675 1

Despite extensive sequence similarity between the N- and C-terminal halves of the Type I isozyme of mammalian hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1), they are functionally distinct, the C-terminal half being responsible for catalysis and the N-terminal half thought to play a regulatory role. We have examined the effects of several site-directed mutations on kinetic and regulatory properties of the rat Type I isozyme. Mutation of the C-terminal residues, Asp 532 to Asn, Arg 539 to Met, and Gly 896 or Gly 898 to Val, resulted in drastic loss of catalytic activity (< 10% of wild-type enzyme), consistent with previous suggestions that these residues are involved in binding of ATP. Mutation of the corresponding residues in the N-terminal half of the enzyme caused much less marked (> 50% of wild type), but significant, effects on activity which are presumed to result from subtle effects on conformation of the enzyme. Mutation of Lys 899 to Met resulted in an approximately 50% decrease in specific activity and an approximately fivefold increase in the Km for ATP, consistent with the view that Lys 899 participates in binding of ATP through electrostatic interactions with the phosphate sidechain. Cys residues corresponding to Cys 158 and Cys 606 of Type I hexokinase are found in other hexokinases that exhibit marked sensitivity to inhibition by the product, glucose 6-phosphate (Glc-6-P), but analogous residues are not found in hexokinases insensitive to Glc-6-P. However, this correlation appears to be coincidental since neither the mutation of Cys 158 or Cys 606 to Ala nor any of the other mutations examined abolished sensitivity of Type I hexokinase to inhibition by the Glc-6-P analog 1,5-anhydroglucitol-6-P or to antagonism of this inhibition by P(i).
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PMID:Residues putatively involved in binding of ATP and glucose 6-phosphate to a mammalian hexokinase: site-directed mutation at analogous positions in the N- and C-terminal halves of the type I isozyme. 764 67

Recent studies have shown that mutations in human beta-cell glucokinase that impair the activity of this key regulatory enzyme of glycolysis can cause early-onset non-insulin-dependent diabetes mellitus (NIDDM). The amino acid sequence of human glucokinase has 31% identity with yeast hexokinase, a related enzyme for which the crystal structure has been determined. This homology has allowed us to model the three-dimensional structure of human glucokinase by analogy to the crystal structure of yeast hexokinase B. This model of human glucokinase provides a basis for understanding the effects of mutations on its enzymatic activity. Residues in the active site and on the surface of the binding cleft for glucose are highly conserved in both enzymes. Regions far from the active site are predicted to differ in conformation, and 10 insertions or deletions that range in size from 1 to 7 residues are located on the protein surface between elements of secondary structure. The model structure suggests that human glucokinase binds glucose in a similar manner to yeast hexokinase. The glucose-binding site contains a conserved aspartic acid, two conserved glutamic acids, and two conserved asparagines that form hydrogen bond interactions with the hydroxyls of the glucose similar to those observed in other sugar-binding proteins. Mutation of residues in the predicted glucose-binding site has been found to greatly reduce enzymatic activity. This model will be useful for future structure/function studies of glucokinase.
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PMID:Molecular model of human beta-cell glucokinase built by analogy to the crystal structure of yeast hexokinase B. 819 64

The structure of the isocitrate dehydrogenase (IDH) complex with bound alpha-ketoglutarate, Ca2+, and NADPH was solved at 2.7-A resolution. The alpha-ketoglutarate binds in the active site at the same position and orientation as isocitrate, with a difference between the two bound molecules of about 0.8 A. The Ca2+ metal is coordinated by alpha-ketoglutarate, three conserved aspartate residues, and a pair of water molecules. The largest motion in the active site relative to the isocitrate enzyme complex is observed for tyrosine 160, which originally forms a hydrogen bond to the labile carboxyl group of isocitrate and moves to form a new hydrogen bond to Asp 307 in the complex with alpha-ketoglutarate. This triggers a number of significant movements among several short loops and adjoining secondary structural elements in the enzyme, most of which participate in dimer stabilization and formation of the active-site cleft. These rearrangements are similar to the ligand-binding-induced movements observed in globins and insulin and serve as a model for an enzymatic mechanism which involves local shifts of secondary structural elements during turnover, rather than large-scale domain closures or loop transitions induced by substrate binding such as those observed in hexokinase or triosephosphate isomerase.
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PMID:Structure of isocitrate dehydrogenase with alpha-ketoglutarate at 2.7-A resolution: conformational changes induced by decarboxylation of isocitrate. 836 1

The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.
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PMID:Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves. 856 28

Rabbit tibialis anterior muscles were stimulated continuously at 10 Hz for periods ranging from 2 min to 96 h and were analyzed for energy reserves and metabolic intermediates. Glycogen, ATP and phosphocreatine fell rapidly during the first 5 min of stimulation. Glycogen continued to fall to very low levels, whereas ATP and phosphocreatine rose, reaching 70% of control by 1 h, despite ongoing stimulation. After 2 h, glycogen also increased, regaining control levels in 4 days. Glucose rose to 4.5 times control in 30 min and still exceeded 2.5 times control at 24 h. In the first 2 min, glycolytic intermediates, glucose 6-phosphate (G-6-P), fructose 1,6-bisphosphate, lactate, and pyruvate more than doubled and then returned to control levels or below. Malate and 3-glycerophosphate rose 600 and 200%, respectively. Both of these compounds participate in shuttling reducing equivalents from cytoplasm into mitochondria. Citrate and alpha-ketoglutarate underwent much more modest changes. Glucose 1,6-bisphosphate (G-1,6-P2) fell to one-third of control by 2 h and then rose dramatically at 4 h. At 4 days it was still twice control. The 6-phosphogluconate (6PG) doubled at 2 min, then rose to 12 times control at 2 h, fell somewhat, and peaked at 16 times control at 24 h. Aspartate and alanine both exhibited a biphasic rise in concentration, whereas glutamate fell to 30% in 15 min and rose slowly after 4 h. The rise in glucose was interpreted to be the consequence of rapid glycogenolysis together with inhibition of hexokinase by G-1,6-P2 and elevated G-6-P. Paradoxically, glycogen resynthesis apparently occurred when the glycogen synthase stimulator, G-6-P, was very low, and the glycolysis stimulator, G-1,6-P2, was high. Although G-1,6-P2 is an inhibitor of 6PG dehydrogenase, the timing of the changes in G-1,6-P2 and 6PG levels suggests that the accumulation of 6PG was initiated by some other influence.
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PMID:Changes in ATP, phosphocreatine, and 16 metabolites in muscle stimulated for up to 96 hours. 889 22


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