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Loranne Agius - One of the best experts on this subject based on the ideXlab platform.
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glucagon induces translocation of Glucokinase from the cytoplasm to the nucleus of hepatocytes by transfer between 6 phosphofructo 2 kinase fructose 2 6 bisphosphatase 2 and the Glucokinase regulatory protein
Biochimica et Biophysica Acta, 2014Co-Authors: Kirsty S Cullen, Loranne Agius, Ziad H Aloanzi, Finbarr Oharte, Catherine ArdenAbstract:Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver Glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating Glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25mM glucose caused decreased binding of Glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of Glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of Glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of Glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in Glucokinase translocation and activity evident in animal models and human diabetes.
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Glucokinase and molecular aspects of liver glycogen metabolism
Biochemical Journal, 2008Co-Authors: Loranne AgiusAbstract:Conversion of glucose into glycogen is a major pathway that contributes to the removal of glucose from the portal vein by the liver in the postprandial state. It is regulated in part by the increase in blood-glucose concentration in the portal vein, which activates Glucokinase, the first enzyme in the pathway, causing an increase in the concentration of glucose 6-P (glucose 6-phosphate), which modulates the phosphorylation state of downstream enzymes by acting synergistically with other allosteric effectors. Glucokinase is regulated by a hierarchy of transcriptional and post-transcriptional mechanisms that are only partially understood. In the fasted state, Glucokinase is in part sequestered in the nucleus in an inactive state, complexed to a specific regulatory protein, GKRP (Glucokinase regulatory protein). This reserve pool is rapidly mobilized to the cytoplasm in the postprandial state in response to an elevated concentration of glucose. The translocation of Glucokinase between the nucleus and cytoplasm is modulated by various metabolic and hormonal conditions. The elevated glucose 6-P concentration, consequent to Glucokinase activation, has a synergistic effect with glucose in promoting dephosphorylation (inactivation) of glycogen phosphorylase and inducing dephosphorylation (activation) of glycogen synthase. The latter involves both a direct ligand-induced conformational change and depletion of the phosphorylated form of glycogen phosphorylase, which is a potent allosteric inhibitor of glycogen synthase phosphatase activity associated with the glycogen-targeting protein, GL [hepatic glycogen-targeting subunit of PP-1 (protein phosphatase-1) encoded by PPP1R3B]. Defects in both the activation of Glucokinase and in the dephosphorylation of glycogen phosphorylase are potential contributing factors to the dysregulation of hepatic glucose metabolism in Type 2 diabetes.
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Glucokinase regulatory protein is associated with mitochondria in hepatocytes
FEBS Letters, 2006Co-Authors: Catherine Arden, Simone Baltrusch, Loranne AgiusAbstract:Abstract The association of Glucokinase with liver mitochondria has been reported [Danial et al. (2003) BAD and Glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424, 952–956]. We confirmed association of Glucokinase immunoreactivity with rat liver mitochondria using Percoll gradient centrifugation and demonstrated its association with the 68 kDa regulatory protein (GKRP) but not with the binding protein phosphofructokinase-2/fructose bisphosphatase-2. Substrates and glucagon induced adaptive changes in the mitochondrial Glucokinase/GKRP ratio suggesting a regulatory role for GKRP. Combined with previous observations that GKRP overexpression partially inhibits glycolysis [de la Iglesia et al. (2000) The role of the regulatory protein of Glucokinase in the glucose sensory mechanism of the hepatocyte. J. Biol. Chem. 275, 10597–10603] these findings suggest that there may be distinct glycolytic pools of Glucokinase.
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dual role of phosphofructokinase 2 fructose bisphosphatase 2 in regulating the compartmentation and expression of Glucokinase in hepatocytes
Diabetes, 2005Co-Authors: Victoria A Payne, Catherine Arden, Alex J Lange, Loranne AgiusAbstract:Hepatic Glucokinase is regulated by a 68-kDa regulatory protein (GKRP) that is both an inhibitor and nuclear receptor for Glucokinase. We tested the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) in regulating Glucokinase compartmentation in hepatocytes. PFK2 catalyzes formation or degradation of the regulator of glycolysis fructose 2,6-bisphosphate (fructose 2,6-P 2 ), depending on its phosphorylation state (ser-32), and is also a Glucokinase-binding protein. Incubation of hepatocytes at 25 mmol/l glucose causes translocation of Glucokinase from the nucleus to the cytoplasm and an increase in fructose 2,6-P 2 . Glucagon caused phosphorylation of PFK2-ser-32, lowered the fructose 2,6-P 2 concentration, and inhibited glucose-induced translocation of Glucokinase. These effects of glucagon were reversed by expression of a kinase-active PFK2 mutant (S32A/H258A) that overrides the suppression of fructose 2,6-P 2 but not by overexpression of wild-type PFK2. Overexpression of PFK2 potentiated Glucokinase expression in hepatocytes transduced with an adenoviral vector–encoding Glucokinase by a mechanism that does not involve stabilization of Glucokinase protein from degradation. It is concluded that PFK2 has a dual role in regulating Glucokinase in hepatocytes: it potentiates Glucokinase protein expression by posttranscriptional mechanisms and favors its cytoplasmic compartmention. Thus, it acts in a complementary mechanism to GKRP, which also regulates Glucokinase protein expression and compartmentation.
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The role of the regulatory protein of Glucokinase in the glucose sensory mechanism of the hepatocyte.
The Journal of biological chemistry, 2000Co-Authors: Núria De La Iglesia, Mohammed H. Mukhtar, Joan J Guinovart, Joan Seoane, Loranne AgiusAbstract:Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic Glucokinase is inhibited by a 68-kDa Glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by Glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased Glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of Glucokinase translocation for glucose and increased the control coefficient of Glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing Glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of Glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, Glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of Glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that Glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to Glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising Glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte.
Jinlei Tang - One of the best experts on this subject based on the ideXlab platform.
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recruiting alternative glucose utilization pathways for improving succinate production
Applied Microbiology and Biotechnology, 2013Co-Authors: Jinlei Tang, Jiao Lu, Hongtao Xu, Xueli ZhangAbstract:The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two Glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS− strains, and their impacts on glucose utilization and succinate production were compared. All PTS− strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS− strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk E. coli (2.13 g/L•h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk E. coli . On the other hand, although Glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L•h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.
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recruiting alternative glucose utilization pathways for improving succinate production
Applied Microbiology and Biotechnology, 2013Co-Authors: Jinlei Tang, Jiao Lu, Hongtao Xu, Xueli ZhangAbstract:The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two Glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS− strains, and their impacts on glucose utilization and succinate production were compared. All PTS− strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS− strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk E. coli (2.13 g/L•h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk E. coli . On the other hand, although Glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L•h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.
E Van Schaftingen - One of the best experts on this subject based on the ideXlab platform.
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Gene expression of Glucokinase regulatory protein in regenerating rat liver
Hepatology, 1997Co-Authors: Jose Luis Rosa, E Van Schaftingen, Michel Detheux, J X Pérez, Ramon BartronsAbstract:The activity and messenger RNA (mRNA) levels of Glucokinase, and the concentration and mRNA levels of its regulatory protein, were analyzed during liver regeneration. The activity of Glucokinase and the concentration of its regulatory protein decreased to 30% and 50%, respectively, after liver resection, remaining low after 1 week. No significant variations in the level of these proteins were found in sham-operated animals. The regulatory protein/Glucokinase molar ratio increased during the replicative phase, to a maximum at 48 hours. The mRNA levels of Glucokinase and of its regulatory protein decreased rapidly after partial hepatectomy to minimum values at 6 hours (15%) and at 12 hours (4%), respectively, returning to normal values at 24 hours and 168 hours, respectively. Sham-operated animals showed a similar decrease in mRNA levels during the prereplicative phase of liver regeneration, suggesting that the initial effects observed in the gene expression of these proteins were due to surgical stress. During the replicative phase, a specific inhibition of the regulatory protein's gene expression was observed in the regenerating liver. A decrease in the content of regulatory protein and the Glucokinase activity, and an increase in the molar ratio of these two proteins correlate with the observed decrease in glycolytic flux, providing further evidence that the phosphorylation of glucose is a control point in the glycolytic/gluconeogenic flux during liver regeneration.
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Effect of mutations on the sensitivity of human beta-cell Glucokinase to liver regulatory protein.
Diabetologia, 1996Co-Authors: Maria Veiga-da-cunha, Y. H. Lee, D. Marotta, Simon J. Pilkis, E Van SchaftingenAbstract:Human beta-cell Glucokinase and its liver counterpart displayed a half-saturating concentration of glucose (S0.5) of about 8 mmol/l and a Hill coefficient of 1.7, and were as sensitive to inhibition by the rat liver regulatory protein as the rat liver enzyme. These results indicate that the N-terminal region of Glucokinase, which differs among these three enzymes, is not implicated in the recognition of the regulatory protein. They also suggest that the regulatory protein, or a related protein, could modulate the affinity of Glucokinase for glucose in beta cells. We have also tested the effect of several mutations, many of which are implicated in maturity onset diabetes of the young. The mutations affected the affinity for glucose and for the regulatory protein to different degrees, indicating that the binding site for these molecules is different. An Asp158 Ala mutation, found in the expression plasmid previously thought to encode the wild-type enzyme, increased the affinity for glucose by about 2.5-fold without changing the affinity for the regulatory protein. The mutations that were found to decrease the affinity for the regulatory protein (Asn166 Arg, Val203 Ala, Asn204 Gln, Lys414 Ala) clustered in the hinge region of Glucokinase and nearby in the large and small domains. These results are in agreement with the concept that part of the binding site for the regulatory protein is situated in the hinge region of this enzyme.
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Heterologous expression of an active rat regulatory protein of Glucokinase
FEBS Letters, 1994Co-Authors: Michel Detheux, E Van SchaftingenAbstract:The cDNA presumed to encode the rat liver regulatory protein of Glucokinase has been expressed in Escherichia coli and a partially soluble protein has been obtained. This recombinant protein was partially purified and found to have the same apparent molecular mass as the regulatory protein purified from rat liver. Like the latter, it inhibited rat liver Glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6-phosphate and fructose 1-phosphate.
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cloning and expression of a xenopus liver cdna encoding a fructose phosphate insensitive regulatory protein of Glucokinase
FEBS Journal, 1994Co-Authors: Maria Veigadacunha, Michel Detheux, Nathalie Watelet, E Van SchaftingenAbstract:Xenopus liver contains a protein inhibitor of Glucokinase that, in contrast to the mammalian regulatory protein of Glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of Glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus Glucokinase with greater affinity than rat Glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.
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Species and tissue distribution of the regulatory protein of Glucokinase.
Biochemical Journal, 1993Co-Authors: Annick Vandercammen, E Van SchaftingenAbstract:Rat liver is known to contain a regulatory protein that inhibits Glucokinase (hexokinase IV or D) competitively versus glucose. This inhibition is greatly reinforced by the presence of fructose 6-phosphate and antagonized by fructose 1-phosphate and by KCl. This protein was now measured in various rat tissues and in the livers of various species by the inhibition it exerts on rat liver Glucokinase. Rat, mouse, rabbit, guinea-pig and pig liver, all of which contain Glucokinase, also contained between 60 and 200 units/g of tissue of a regulatory protein displaying the properties mentioned above. By contrast, this protein could not be detected in cat, goat, chicken or trout liver, or in rat brain, heart, skeletal muscle, kidney and spleen, all tissues from which Glucokinase is missing. Fructose 1-phosphate stimulated Glucokinase in extracts of human liver, indicating the presence of regulatory protein. In addition, antibodies raised against rat regulatory protein allowed the detection of an approximately 60 kDa polypeptide in rat, guinea pig, rabbit and human liver. The livers of the toad Bufo marinus, of Xenopus laevis and of the turtle Pseudemys scripta elegans contained a regulatory protein similar to that of the rat, with, however, the major difference that it was not sensitive to fructose 6-phosphate or fructose 1-phosphate. In rat liver, the regulatory protein was detectable 4 days before birth. Its concentration increased afterwards to reach the adult level at day 30 of extrauterine life, whereas Glucokinase only appeared after day 15. In the liver of the adult rat, starvation and streptozotocin-diabetes caused a 50-60% decrease in the concentration of regulatory protein after 7 days, whereas Glucokinase activity fell to about 20% of its initial level. When 4-day-starved rats were refed, or when diabetic rats were treated with insulin, the concentration of regulatory protein slowly increased to reach about 85% of the control level after 3 days, whereas the Glucokinase activity was normalized after the same delay. The fact that there appears to be no situation in which Glucokinase is expressed without regulatory protein is in agreement with the notion that the regulatory protein forms a functional entity with this enzyme.
Xueli Zhang - One of the best experts on this subject based on the ideXlab platform.
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recruiting alternative glucose utilization pathways for improving succinate production
Applied Microbiology and Biotechnology, 2013Co-Authors: Jinlei Tang, Jiao Lu, Hongtao Xu, Xueli ZhangAbstract:The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two Glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS− strains, and their impacts on glucose utilization and succinate production were compared. All PTS− strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS− strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk E. coli (2.13 g/L•h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk E. coli . On the other hand, although Glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L•h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.
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recruiting alternative glucose utilization pathways for improving succinate production
Applied Microbiology and Biotechnology, 2013Co-Authors: Jinlei Tang, Jiao Lu, Hongtao Xu, Xueli ZhangAbstract:The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two Glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS− strains, and their impacts on glucose utilization and succinate production were compared. All PTS− strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS− strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk E. coli (2.13 g/L•h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk E. coli . On the other hand, although Glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L•h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.
Victoria Guixé - One of the best experts on this subject based on the ideXlab platform.
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adp dependent phosphofructokinases from the archaeal order methanosarcinales display redundant Glucokinase activity
Archives of Biochemistry and Biophysics, 2017Co-Authors: Ricardo A. Zamora, Felipe Gonzalezordenes, Victor Castrofernandez, Victoria GuixéAbstract:The genome of Methanosarcinales organisms presents both ADP-dependent Glucokinase and phosphofructokinase genes. However, Methanococcoides burtonii has a truncate Glucokinase gene with a large deletion at the C-terminal, where the catalytic GXGD motif is located. Characterization of its phosphofructokinase annotated protein shows that is a bifunctional enzyme able to supply the absence of the Glucokinase activity. Moreover, kinetic analyses of the phosphofructokinase annotated enzyme from, Methanohalobium evestigatum demonstrated that this enzyme is also bifunctional. The high conservation of the active site residues of all the enzymes from the order Methanosarcinales suggest that they should be bifunctional, as was previously reported for the ADP-dependent kinases from Methanococcales, highlighting the redundancy of the Glucokinase activity in this archaeal group. The presence of active glycolytic enzymes would be important when glycogen storage of these organisms needs to be degraded to be used as energy source. Kinetic and structural information allows us to establish a substrate specificity signature that identifies specific GK or PFK, and bifunctional enzymes in this family.
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crystal structure saxs and kinetic mechanism of hyperthermophilic adp dependent Glucokinase from thermococcus litoralis reveal a conserved mechanism for catalysis
PLOS ONE, 2013Co-Authors: Jaime Andres Rivaspardo, Victor Castrofernandez, Alejandra Herreramorande, Francisco J Fernandez, Cristina M Vega, Victoria GuixéAbstract:ADP-dependent Glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent Glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the Glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.