Inhibition by Glucose 6-Phosphate

Total Page:16

File Type:pdf, Size:1020Kb

Inhibition by Glucose 6-Phosphate Proc. Nati. Acad. Sci. USA Vol. 82, pp. 1552-1554, March 1985 Medical Sciences High glucose concentrations partially release hexokinase from inhibition by glucose 6-phosphate (diabetes/kinetics) SHINYA FumII AND ERNEST BEUTLER* Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi, 755, Japan; and Scripps Clinic and Research Foundation, Department of Basic and Clinical Research, 10666 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Ernest Beutler, November 2, 1984 ABSTRACT The phosphorylation of glucose by human cose concentrations, and although previous reports showed erythrocyte hexokinase follows classical Michaelis-Menten ki- no competition of these inhibitors with glucose, no details netics; hexokinase manifests maximum activity at 5 mM glu- were presented. In the present studies, we extensively ex- cose, and no further increase in activity can be measured at amined the velocity of the hexokinase reaction in the pres- higher glucose concentrations. However, the erythrocytes of ence of inhibitors and high concentrations of glucose in or- diabetics and normal erythrocytes incubated with high concen- der to shed light on the cause of the elevation of glucose 6- trations of glucose contain increased concentrations of glucose phosphate levels in the erythrocytes of diabetics. 6-phosphate. To elucidate the mechanism of accumulation of glucose 6-phosphate when erythrocytes are exposed to high glucose concentrations, hexokinase activity was examined in EXPERIMENTAL PROCEDURES the presence of naturally occurring inhibitors, such as glucose Partial Purification of Hexinas from Human Erythro- 1,6-bisphosphate, 2,3-diphosphoglycerate, ADP, and glucose cytes. Hexokinase was partially purified by a modification of 6-phosphate at physiological concentrations. Without inhibi- the method of Rijksen and Staal (9). Three hundred millili- tors or in the presence ofglucose 1,6-bisphosphate, 2,3-diphos- ters of blood was freed of leukocytes and platelets by pass- phoglycerate, and ADP, maximum hexokinase activity was ob- ing through a cellulose column (10), and the erythrocytes served at 5 mM glucose concentration. On the contrary, in the were washed 3 times with isotonic saline. The packed eryth- presence of glucose 6-phosphate, hexokinase activity increased rocytes were lysed with 1 vol of 0.4% saponin in H20 for 1 at glucose concentrations >5 mM; inhibition by glucose 6- hr. The hemolysate was mixed with 0.5 vol of 501% suspen- phosphate was partially competitive with glucose. The reliefby sion of DEAE-cellulose (DE52) equilibrated with 10 mM po- glucose of glucose 6-phosphate inhibition of hexokinase is a tassium phosphate buffer (pH 7.3) containing 3 mM 2-mer- possible explanation of the increased glucose 6-phosphate level captoethanol, and 3 mM NaF. After washing with the same in diabetic erythrocytes. buffer, the enzyme was eluted with 500 mM potassium phos- phate buffer (pH 7.3) containing 3 mM 2-mercaptoethanol, 3 The erythrocytes of diabetic patients contain increased lev- mM NaF, and 10% (NH4)2SO4. Solid ammonium sulfate was els of the glycohemoglobin hemoglobin Al. The concentra- added to the enzyme solution to 75% saturation. After stand- tion of glucose 6-phosphate has been found to be increased ing overnight at 4TC the precipitate was collected by centrifu- in the erythrocytes of diabetic patients (1-3), and it has been gation, was dissolved in 10 mM potassium phosphate buffer suggested that glucose 6-phosphate may be the precursor of (pH 7.3) containing 3 mM 2-mercaptoethanol and 3 mM the carbohydrate variety of this glycohemoglobin. The NaF, and was dialyzed against the same buffer. The dialyzed mechanism by which erythrocyte glucose 6-phosphate levels solution was applied to a DEAE-cellulose column (0.8 x 23 become elevated in diabetics is by no means obvious, how- cm) equilibrated with the same buffer. The column was ever. The human erythrocyte is highly permeable to glucose. washed with the same buffer and then with the same buffer Human erythrocyte hexokinase has a very low Km for glu- containing 10 mM KCN. Elution of the enzyme was per- cose (50-80 x 10-6 M), and it is saturated with glucose even formed with a 500-ml linear gradient of0-500 mM KCI in the at normal plasma glucose levels (5 mM). Accordingly, the same buffer containing 10 mM KCN. Fractions of 5.5 ml increase in glucose 6-phosphate concentration in diabetic were collected and assayed for hexokinase, glucose-6-phos- erythrocytes is obviously not merely the direct result of in- phate dehydrogenase, glucose-phosphate isomerase, and creased availability of glucose to hexokinase. adenylate kinase (11). The fractions containing hexokinase It has been proposed that acidemia due to ketoacidosis activity and free ofglucose-6phosphate dehydrogenase, glu- sometimes seen in severe or untreated diabetics might cause cose phosphate isomerase, and adenylate kinase were elevation of glucose 6-phosphate levels in the erythrocytes pooled and concentrated by ultrafiltration. The enzyme was of diabetics, because phosphofructokinase activity is inhibit- stored at -20TC in 10 mM potassium phosphate buffer (pH ed by the lower pH (4). However, most diabetics are not 7.0) containing 3 mM 2-mercaptoethanol, 3 mM NaF, 2 mM acidotic. Moreover, Stevens et al. (1) and Tegos and Beutler glucose, and 10%6 (vol/vol) glycerol. Before use, the stored (3) observed no crossover in the levels of erythrocyte glyco- enzyme was dialyzed against 10 mM Tris HC1 (pH 8.0). lytic intermediates in diabetics. This suggested that high glu- Hexokinase Assay. The standard assay mixture (11) con- cose concentrations affected the hexokinase reaction itself. tained 50 mM Tris HCI, pH 8.0/0.2 mM NADP/1 mM Hexokinase normally operates in a state of partial inhibi- ATP/5 mM MgCl2/0.1 international unit (IU) of glucose-6- tion by some glycolytic intermediates (5-8). Relief of inhibi- phosphate dehydrogenase and glucose at the concentrations tion of hexokinase by high concentrations of glucose would indicated in the text. Absorbance was measured at 340 nm at result in the apparent activation of the enzyme by high glu- 37°C for 10-20 min. When glucose 6-phosvhate was studied as an inhibitor, it The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: IU, international unit(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1552 Downloaded by guest on September 28, 2021 Medical Sciences: Fujii and Beutler Proc. NatL Acad. Sci. USA 82 (1985) 1553 was necessary to measure hexokinase activity in another Studies with Purified Erythrocyte Hexokinase. Fig. 1 shows system in which ADP production was determined in the py- the effect of high concentrations of glucose on the inhibition ruvate kinase reaction. This assay system contained 50 mM of hexokinase by glucose 6-phosphate using the PK-LDH as- Tris HCl, pH 8.0/5 mM MgCl2/75 mM KCl/1 mM ATP/2 say method. Without glucose 6-phosphate, hexokinase activ- mM phosphoenolpyruvate/0.2 mM NADH/3 IU ofpyruvate ity was maximal at 5 mM glucose and 87.6% ± 1.5% (mean kinase/3 IU of lactate dehydrogenase. The reaction was fol- ± SEM) of the Vma at 0.5 mM glucose; glucose concentra- lowed by measuring optical density at 340 nm at 370C for 3 tions >5 mM resulted in no increase of activity. Since the min. One unit of enzyme activity was defined as the amount Km for glucose is 50-80 x 10-6 M, these findings were in of enzyme that catalyzes the formation of 1 umol of glucose perfect agreement with the predicted value from the Michae- 6-phosphate or 1 ,umol of ADP per min. In both systems, the lis-Menten equation, indicating the accuracy of the method reaction was started by adding 0.004 unit of hexokinase per used here. It is clear that hexokinase is completely saturated ml ofreaction mixture. A cuvette without hexokinase served at 5 mM glucose. In the presence of glucose 6-phosphate, on as the blank. the contrary, the activity is slightly, but significantly in- creased at glucose concentrations >5 mM. These results in- RESULTS dicate that hexokinase inhibition by glucose 6-phosphate is partially relieved by high concentrations of glucose. The Glucose 6-Phosphate Level After Incubation of Human data have been replotted in the inserted figure to provide Erythrocytes with Glucose. Fasting blood was drawn into insight into the relationship between glucose concentration heparin from normal volunteers. After centrifugation at 2000 and inhibition by glucose 6-phosphate. The curves obtained x g for 10 min, packed erythrocytes were suspended in 1 vol suggest the presence of a mixed type of inhibition (12). of 0.1 M Hepes buffer (pH 7.4) containing 135 mM NaCl in Using the standard assay, hexokinase activity in the ab- the presence of 5 mM and 30 mM glucose. Incubation was sence and presence of glucose 1,6-bisphosphate, 2,3-diphos- continued for 1 hr at 37°C in a shaking bath. The pH of both phoglycerate, and ADP at various glucose concentrations suspensions was the same at the end of the incubation. The was examined (data not given). In the absence of inhibitors, reaction was terminated by adding 4 vol of ice-cold 4% per- hexokinase activity reached its maximum activity at 5 mM chloric acid. The concentration of glucose 6-phosphate was glucose; higher glucose concentration resulted in no increase measured as described (11). In eight separate experiments, of activity. In the presence of physiological concentrations the glucose 6-phosphate concentration in the presence of 5 of glucose 1,6-bisphosphate (200 ,uM), 2,3-diphosphoglycer- mM glucose was found to be 30.19 ± 0.88 ,umol per liter of ate (5 mM), and ADP (1 mM), hexokinase activity also pla- packed erythrocytes (mean ± SEM); in the presence of 30 teaued at 5 mM and higher glucose concentrations, indicat- mM glucose, the glucose 6-phosphate concentration was ing that a high concentration of glucose is not competitive 35.8 ± 1.58 ,uM.
Recommended publications
  • Galactokinase (B) Glucokinase (C) Galactose-1-Phosphate Uridyltransferase (D) UDP-Galactose 4- Epimerase Sol
    1. Which of the following enzymes are not involved in galactose metabolism? (a) Galactokinase (b) Glucokinase (c) Galactose-1-Phosphate Uridyltransferase (d) UDP-Galactose 4- epimerase Sol. (b) Glucokinase. 2. Which of the following enzymes leads to a glycogen storage disease known as Tarui’s disease? (a) Glucokinase (b) Pyruvate Kinase (c) Phosphofructokinase (d) Phosphoglucomutase Sol. (c) Phosphofructokinase. 3. Which of the following enzymes is defective in galactosemia- a fatal genetic disorder in infants? (a) Glucokinase (b) Galactokinase (c) UDP-Galactose 4- epimerase (d) Galactose-1-Phosphate Uridyltransferase Sol. (d) Galactose-1-Phosphate Uridyltransferase. 4. Which of the following enzyme deficiency leads to hemolytic anaemia? (a) Glucokinase (b) Pyruvate Kinase (c) Phosphoglucomutase (d) Phosphofructokinase Sol. (b) Pyruvate Kinase. 5. Which of the following glucose transporters are important in fructose transport in the intestine? (a) GLUT5 (b) GLUT3 (c) GLUT4 (d) GLUT7 Sol. (a) GLUT5. 6. Which of the following is a tricarboxylic acid? (a) Acetic acid (b) Succinic acid (c) Oxaloacetic acid (d) Citric acid Sol.(d) Citric acid. 7. Which of the following enzymes plays an important role in tumour metabolism? (a) Glucokinase (b) Pyruvate Kinase M2 (c) Phosphoglucomutase (d) Phosphofructokinase Sol. (b) Pyruvate Kinase M2. 8. Which of the following metabolites negatively regulates pyruvate kinase? 1. (a) Citrate (b) Alanine (c) Acetyl CoA (d) Fructose-1,6-Bisphosphate Sol. (b) Alanine 9. The glycerol phosphate shuttle functions in___________. (a) Lipid catabolism (b) Triglyceride synthesis (c) Anaerobic glycolysis for the regeneration of NAD (d) Aerobic glycolysis to transport NADH equivalents resulting from glycolysis into mitochondria. Sol. (d) Aerobic glycolysis to transport NADH equivalents resulting from glycolysis into mitochondria.
    [Show full text]
  • Table S1. List of Oligonucleotide Primers Used
    Table S1. List of oligonucleotide primers used. Cla4 LF-5' GTAGGATCCGCTCTGTCAAGCCTCCGACC M629Arev CCTCCCTCCATGTACTCcgcGATGACCCAgAGCTCGTTG M629Afwd CAACGAGCTcTGGGTCATCgcgGAGTACATGGAGGGAGG LF-3' GTAGGCCATCTAGGCCGCAATCTCGTCAAGTAAAGTCG RF-5' GTAGGCCTGAGTGGCCCGAGATTGCAACGTGTAACC RF-3' GTAGGATCCCGTACGCTGCGATCGCTTGC Ukc1 LF-5' GCAATATTATGTCTACTTTGAGCG M398Arev CCGCCGGGCAAgAAtTCcgcGAGAAGGTACAGATACGc M398Afwd gCGTATCTGTACCTTCTCgcgGAaTTcTTGCCCGGCGG LF-3' GAGGCCATCTAGGCCATTTACGATGGCAGACAAAGG RF-5' GTGGCCTGAGTGGCCATTGGTTTGGGCGAATGGC RF-3' GCAATATTCGTACGTCAACAGCGCG Nrc2 LF-5' GCAATATTTCGAAAAGGGTCGTTCC M454Grev GCCACCCATGCAGTAcTCgccGCAGAGGTAGAGGTAATC M454Gfwd GATTACCTCTACCTCTGCggcGAgTACTGCATGGGTGGC LF-3' GAGGCCATCTAGGCCGACGAGTGAAGCTTTCGAGCG RF-5' GAGGCCTGAGTGGCCTAAGCATCTTGGCTTCTGC RF-3' GCAATATTCGGTCAACGCTTTTCAGATACC Ipl1 LF-5' GTCAATATTCTACTTTGTGAAGACGCTGC M629Arev GCTCCCCACGACCAGCgAATTCGATagcGAGGAAGACTCGGCCCTCATC M629Afwd GATGAGGGCCGAGTCTTCCTCgctATCGAATTcGCTGGTCGTGGGGAGC LF-3' TGAGGCCATCTAGGCCGGTGCCTTAGATTCCGTATAGC RF-5' CATGGCCTGAGTGGCCGATTCTTCTTCTGTCATCGAC RF-3' GACAATATTGCTGACCTTGTCTACTTGG Ire1 LF-5' GCAATATTAAAGCACAACTCAACGC D1014Arev CCGTAGCCAAGCACCTCGgCCGAtATcGTGAGCGAAG D1014Afwd CTTCGCTCACgATaTCGGcCGAGGTGCTTGGCTACGG LF-3' GAGGCCATCTAGGCCAACTGGGCAAAGGAGATGGA RF-5' GAGGCCTGAGTGGCCGTGCGCCTGTGTATCTCTTTG RF-3' GCAATATTGGCCATCTGAGGGCTGAC Kin28 LF-5' GACAATATTCATCTTTCACCCTTCCAAAG L94Arev TGATGAGTGCTTCTAGATTGGTGTCggcGAAcTCgAGCACCAGGTTG L94Afwd CAACCTGGTGCTcGAgTTCgccGACACCAATCTAGAAGCACTCATCA LF-3' TGAGGCCATCTAGGCCCACAGAGATCCGCTTTAATGC RF-5' CATGGCCTGAGTGGCCAGGGCTAGTACGACCTCG
    [Show full text]
  • Pyruvate-Phosphate Dikinase of Oxymonads and Parabasalia and the Evolution of Pyrophosphate-Dependent Glycolysis in Anaerobic Eukaryotes† Claudio H
    EUKARYOTIC CELL, Jan. 2006, p. 148–154 Vol. 5, No. 1 1535-9778/06/$08.00ϩ0 doi:10.1128/EC.5.1.148–154.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Pyruvate-Phosphate Dikinase of Oxymonads and Parabasalia and the Evolution of Pyrophosphate-Dependent Glycolysis in Anaerobic Eukaryotes† Claudio H. Slamovits and Patrick J. Keeling* Canadian Institute for Advanced Research, Botany Department, University of British Columbia, 3529-6270 University Boulevard, Vancouver, British Columbia V6T 1Z4, Canada Received 29 September 2005/Accepted 8 November 2005 In pyrophosphate-dependent glycolysis, the ATP/ADP-dependent enzymes phosphofructokinase (PFK) and pyruvate kinase are replaced by the pyrophosphate-dependent PFK and pyruvate phosphate dikinase (PPDK), respectively. This variant of glycolysis is widespread among bacteria, but it also occurs in a few parasitic anaerobic eukaryotes such as Giardia and Entamoeba spp. We sequenced two genes for PPDK from the amitochondriate oxymonad Streblomastix strix and found evidence for PPDK in Trichomonas vaginalis and other parabasalia, where this enzyme was thought to be absent. The Streblomastix and Giardia genes may be related to one another, but those of Entamoeba and perhaps Trichomonas are distinct and more closely related to bacterial homologues. These findings suggest that pyrophosphate-dependent glycolysis is more widespread in eukaryotes than previously thought, enzymes from the pathway coexists with ATP-dependent more often than previously thought and may be spread by lateral transfer of genes for pyrophosphate-dependent enzymes from bacteria. Adaptation to anaerobic metabolism is a complex process (PPDK), respectively (for a comparison of these reactions, see involving changes to many proteins and pathways of critical reference 21).
    [Show full text]
  • Pyrophosphate-Dependent Enzymes in Walled Bacteria Phylogenetically Related to the Wall-Less Bacteria of the Class Mollicutes?
    INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1989, p. 413419 Vol. 39, No. 4 OO20-7713/89/O4O413-07$02.00/0 Copyright 0 1989, International Union of Microbiological Societies Pyrophosphate-Dependent Enzymes in Walled Bacteria Phylogenetically Related to the Wall-Less Bacteria of the Class Mollicutes? JAMES P. PETZEL,'S PAUL A. HARTMAN,'* AND MILTON J. ALLISON2 Department of Microbiology, Iowa State University, Ames, Iowa 5001 1-321I ,I and National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 500102 Some of the wall-less bacteria of the class Mollicutes (mycoplasmas) have pyrophosphate (PP,)-dependent enzymic activities, including PP,-dependent phosphofructokinase (PP,-PFK), PP,-dependent nucleoside kinase, and pyruvate,orthophosphate dikinase (PPDK) activities. In most other bacteria, adenosine 5'-triphosphate (ATP), not PP,, is the cofactor of analogous enzymic reactions. Because PP,-dependent enzymes are more common among mollicutes than other bacteria, we describe here an examination of the six walled bacteria that have been reported to be phylogenetically related to the mollicutes (Clostridium innocuum, Clostridium ramosum, Erysipelothrix rhusiopathiae, Lactobacillus catenaformis, Lactobacillus vitulinus, and Streptococcus pleomorphus) for PP,-PFK, ATP-dependent PFK, phosphoenolpyruvate carboxytransphosphorylase, PPDK, and PP,- and ATP-dependent acetate kinases. Two anaerobic mollicutes, Anaeroplasma intermedium and Asteroleplasma anuerobium, were also tested. C. innocuum, E. rhusiopathiue, S. pleomorphus, and Anuero- plasma intermedium had PPi-PFK activities, whereas C. ramosum, the two lactobacilli, and Asteroleplasma anaerobium had only ATP-dependent PFK activities. Asteroleplasma anaerobium and all of the walled bacteria except E. rhusiopathiue had PPDK activities. All of the species except Asteroleplasma anaerobium and E. rhusiopathiae also had pyruvate kinase activities; the effects of allosteric activators were tested.
    [Show full text]
  • Regulation of Fructose-6-Phosphate 2-Kinase By
    Proc. Natt Acad. Sci. USA Vol. 79, pp. 325-329, January 1982 Biochemistry Regulation of fructose-6-phosphate 2-kinase by phosphorylation and dephosphorylation: Possible mechanism for coordinated control of glycolysis and glycogenolysis (phosphofructokinase) EISUKE FURUYA*, MOTOKO YOKOYAMA, AND KOSAKU UYEDAt Pre-Clinical Science Unit of the Veterans Administration Medical Center, 4500 South Lancaster Road, Dallas, Texas 75216; and Biochemistry Department of the University ofTexas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235 Communicated by Jesse C. Rabinowitz, September 28, 1981 ABSTRACT The kinetic properties and the control mecha- Fructose 6-phosphate + ATP nism of fructose-6-phosphate 2-kinase (ATP: D-fructose-6-phos- -3 Fructose + ADP. [1] phate 2-phosphotransferase) were investigated. The molecular 2,6-bisphosphate weight of the enzyme is -100,000 as determined by gel filtration. The plot of initial velocity versus ATP concentration is hyperbolic We have shown that the administration of extremely low con- with a K. of 1.2 mM. However, the plot of enzyme activity as a centrations of glucagon (0.1 fM) or high concentrations of epi- function of fructose 6-phosphate is sigmoidal. The apparent K0.5 nephrine (10 ,uM) to hepatocytes results in inactivation offruc- for fructose 6-phosphate is 20 ,IM. Fructose-6-phosphate 2-kinase tose-6-phosphate 2-kinase and concomitant decrease in the is inactivated by -the catalytic subunit of cyclic AMP-dependent fructose 2,6-bisphosphate level (12). These results, as well as protein kinase, and the inactivation is closely correlated with phos- more recent data using Ca2+ and the Ca2+ ionophore A23187 phorylation.
    [Show full text]
  • RNA Helicases in RNA Decay
    Biochemical Society Transactions (2018) 46 163–172 https://doi.org/10.1042/BST20170052 Review Article RNA helicases in RNA decay Vanessa Khemici and Patrick Linder Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland Correspondence: Patrick Linder ([email protected]) RNA molecules have the tendency to fold into complex structures or to associate with complementary RNAs that exoribonucleases have difficulties processing or degrading. Therefore, degradosomes in bacteria and organelles as well as exosomes in eukaryotes have teamed-up with RNA helicases. Whereas bacterial degradosomes are associated with RNA helicases from the DEAD-box family, the exosomes and mitochondrial degra- dosome use the help of Ski2-like and Suv3 RNA helicases. Introduction All living cells encounter situations where they need to adapt gene expression to changing environ- mental conditions. The synthesis of new mRNAs to be used for translation and the release of seques- tered or translational inactive mRNAs allow the cells to express new proteins. On the other hand, processing and degradation of RNAs not only helps to recycle essential components, but also to shut down expression of genes that are no longer required or would even be detrimental for living under a new condition. Moreover, remnants of processed or aberrant transcripts must rapidly be degraded to avoid the production of useless or even toxic peptides and proteins. Eubacteria, Archaea, and eukar- yotes have developed dedicated pathways and complexes to process RNA, check the accuracy of RNAs (surveillance), and feed undesired RNA into exoribonucleases that degrade RNA in a 30–50 or 50–30 direction.
    [Show full text]
  • Glycolysis and Gluconeogenesis Are Regulated Independently
    Substrate cycles in glucose metabolism Glycolysis and gluconeogenesis! are regulated independently (the ΔG! values shown are for the corresponding! reactions in liver; in kJ/mol). All six! reactions are exergonic.! Cellular [F2,6BP] depends on the balance between its! rates of synthesis and degradation by PFK-2 ! (phosphofructokinase-2) and FBPase-2 (fructose bisphosphatase-2).! These activities are located on different domains of the! same homodimeric protein (a bifunctional enzyme).! The bifunctional enzyme is regulated by allosteric effectors and by! phosphorylation/dephosphorylation catalyzed by PKA (protein! kinase A) and a phosphoprotein phosphatase.! F2,6BP activates PFK-1 and inhibits FBPase-1. When blood [glucose] is high, cAMP levels decrease, and [F2,6BP]! rises, promoting glycolysis.! The F2,6BP control system in muscle differs from that in liver.! Hormones that stimulate glycogen breakdown in heart muscle lead to phosphorylation of the bifunctional enzyme that stimulates rather than inhibits PFK-2. The increasing [F2,6BP] stimulates glycolysis so that glycogen breakdown and glycolysis are coordinated.! The skeletal muscle PFK-2/PBPase-2 isozyme lacks a phosphorylation! site and is thus not subject to cAMP-dependent control.! Alanine inhibits pyruvate kinase.! Alanine, a major gluconeogenic precursor, inhibits PK.! Liver PK is also inactivated by phosphorylation. Phosphorylation! activates glycogen phosphorylase and FBPase-2: thus the pathways of ! gluconeogenesis and glycogen breakdown both flow towards G6P, ! which is converted to glucose for export from the liver.! Hexokinase/glucokinase and G6Pase activities are also controlled.! Glucose metabolism is regulated by long-term changes in the amounts of enzymes synthesized.! Rates of transcription and mRNA stabilities encoding regulatory enzymes are influenced by hormones.
    [Show full text]
  • Crispri-Library-Guided Target Identification for Engineering
    microorganisms Article CRISPRi-Library-Guided Target Identification for Engineering Carotenoid Production by Corynebacterium glutamicum Vanessa L. Göttl, Ina Schmitt, Kristina Braun, Petra Peters-Wendisch, Volker F. Wendisch * and Nadja A. Henke Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, 33615 Bielefeld, Germany; [email protected] (V.L.G.); [email protected] (I.S.); [email protected] (K.B.); [email protected] (P.P.-W.); [email protected] (N.A.H.) * Correspondence: [email protected]; Tel.: +49-521-106-5611 Abstract: Corynebacterium glutamicum is a prominent production host for various value-added com- pounds in white biotechnology. Gene repression by dCas9/clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) allows for the identification of target genes for metabolic engineering. In this study, a CRISPRi-based library for the repression of 74 genes of C. glutamicum was constructed. The chosen genes included genes encoding enzymes of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, regulatory genes, as well as genes of the methylerythritol phosphate and carotenoid biosynthesis pathways. As expected, CRISPRi-mediated repression of the carotenogenesis repressor gene crtR resulted in increased pigmentation and cellular content of the native carotenoid pigment decaprenoxanthin. CRISPRi screening identified 14 genes that affected decaprenoxanthin biosynthesis when repressed. Carotenoid biosynthesis was signifi- Citation: Göttl, V.L.; Schmitt, I.; cantly decreased upon CRISPRi-mediated repression of 11 of these genes, while repression of 3 genes Braun, K.; Peters-Wendisch, P.; was beneficial for decaprenoxanthin production. Largely, but not in all cases, deletion of selected Wendisch, V.F.; Henke, N.A.
    [Show full text]
  • Carbohydrate Kinases: a Conserved Mechanism Across Differing Folds
    catalysts Review Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds Sumita Roy 1, Mirella Vivoli Vega 2 and Nicholas J. Harmer 1,* 1 Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK; [email protected] 2 Department of Biomedical Experimental and Clinical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy; mirella.vivoli@unifi.it * Correspondence: [email protected]; Tel.: +44-1392-725179 Received: 2 November 2018; Accepted: 21 December 2018; Published: 2 January 2019 Abstract: Carbohydrate kinases activate a wide variety of monosaccharides by adding a phosphate group, usually from ATP. This modification is fundamental to saccharide utilization, and it is likely a very ancient reaction. Modern organisms contain carbohydrate kinases from at least five main protein families. These range from the highly specialized inositol kinases, to the ribokinases and galactokinases, which belong to families that phosphorylate a wide range of substrates. The carbohydrate kinases utilize a common strategy to drive the reaction between the sugar hydroxyl and the donor phosphate. Each sugar is held in position by a network of hydrogen bonds to the non-reactive hydroxyls (and other functional groups). The reactive hydroxyl is deprotonated, usually by an aspartic acid side chain acting as a catalytic base. The deprotonated hydroxyl then attacks the donor phosphate. The resulting pentacoordinate transition state is stabilized by an adjacent divalent cation, and sometimes by a positively charged protein side chain or the presence of an anion hole. Many carbohydrate kinases are allosterically regulated using a wide variety of strategies, due to their roles at critical control points in carbohydrate metabolism.
    [Show full text]
  • Metabolic Labelling of Bacterial Isoprenoids Produced by the Methylerythritol Phosphate Pathway : a Starting Point Towards a New Inhibitor
    UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES CHIMIQUES UMR 7177 & 7199 THÈSE présentée par : Zoljargal BAATARKHUU soutenue le : 05 Septembre 2017 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Chimie Bio-Organique Metabolic labelling of bacterial isoprenoids produced by the Methylerythritol phosphate pathway : A starting point towards a new inhibitor THÈSE dirigée par : Dr Alain WAGNER Directeur de recherche, CNRS Dr Myriam SEEMANN Directeur de recherche, CNRS RAPPORTEURS : Dr Joelle DUBOIS Directeur de recherche, Institut de Chimie des Substances Naturelles, Paris Dr Alain BURGER Professeur, Université de Nice, Nice (In memory of my beloved memory of my (In father Baatarkhuu Dugajii) Résumé de thèse en français 1) Introduction Les isopréonoïdes forment une famille de produits naturels parmi les plus diverses avec plus de 50000 composés connus. 1 Ils sont présents dans tous les organismes vivants. Ils ont de nombreux rôles biologiques, allant du transport d’électrons, à la biosynthèse des membranes cellulaires. Malgré la diversité des isoprénoïdes, ils sont synthétisés à partir de deux précurseurs : le diphosphate d’isopentényle (IPP) et le diphosphate de diméthylallyle (DMAPP). Deux voies de biosynthèse existent pour la formation de ces molécules : la voie du mévalonate et la voie du méthylérythritol phosphate (MEP), découverte plus récemment. 2, 3 Cette dernière voie de synthèse est utilisée par les micro-organismes dont des pathogènes comme Mycobacterium tuberculosis (bactérie responsable de la tuberculose), Vibrio cholerae (bactérie responsable du choléra) et Plasmodium falciparum (parasite responsable de la malaria). Elle est cependant absente chez l’humain et, par conséquent, est une cible de choix pour le développement d’un nouveau médicament antibactérien ou antiparasitaire.
    [Show full text]
  • PFKM Gene Phosphofructokinase, Muscle
    PFKM gene phosphofructokinase, muscle Normal Function The PFKM gene provides instructions for making one piece (the PFKM subunit) of an enzyme called phosphofructokinase. This enzyme plays a role in the breakdown of a complex sugar called glycogen, which is a major source of stored energy in the body. The phosphofructokinase enzyme is made up of four subunits and is found in a variety of tissues. Different combinations of subunits are found in different tissues. In muscles used for movement (skeletal muscles), the phosphofructokinase enzyme is composed solely of subunits produced from the PFKM gene. The cells' main source of energy is stored as glycogen. Glycogen can be broken down rapidly into the simple sugar glucose when energy is needed, for instance to maintain normal blood sugar levels between meals or for energy during exercise. Phosphofructokinase composed of PFKM subunits is involved in the sequence of events that breaks down glycogen to provide energy to muscle cells. Specifically, the enzyme converts a molecule called fructose-6-phosphate to a molecule called fructose 1,6-bisphosphate. Health Conditions Related to Genetic Changes Glycogen storage disease type VII At least 20 mutations in the PFKM gene have been found to cause glycogen storage disease type VII (GSDVII). This condition is characterized by an inability to break down glycogen in muscle cells, resulting in muscle cramps and weakness that can vary in severity among affected individuals. PFKM gene mutations that cause GSDVII result in the production of PFKM subunits that have little or no function. One PFKM gene mutation accounts for most cases of GSDVII in people with Ashkenazi Jewish ancestry.
    [Show full text]
  • Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape During in Vitro Culture1[OPEN]
    Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture1[OPEN] Jörg Schwender, Inga Hebbelmann, Nicolas Heinzel, Tatjana Hildebrandt, Alistair Rogers, Dhiraj Naik, Matthias Klapperstück, Hans-Peter Braun, Falk Schreiber, Peter Denolf, Ljudmilla Borisjuk, and Hardy Rolletschek* Brookhaven National Laboratory, Biological, Environmental, and Climate Sciences Department, Upton, New York11973(J.S.,I.H.,A.R.,D.N.);DepartmentofMolecularGenetics,LeibnizInstituteofPlantGenetics and Crop Plant Research, D–06466 Gatersleben, Germany (N.H., L.B., H.R.); Institut für Pflanzengenetik, Universität Hannover, 30419 Hannover, Germany (T.H., H.-P.B.); Department of Environmental Science, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India (D.N.); Clayton School of Information Technology, Monash University, Melbourne, Victoria 3800, Australia (M.K., F.S.); Institute of Computer Science, University Halle-Wittenberg, 06120 Halle, Germany (F.S.); and Bayer CropScience, 9052 Zwijnaarde, Belgium (P.D.) ORCID IDs: 0000-0002-1226-2337 (D.N.); 0000-0002-4459-9727 (H.-P.B.); 0000-0003-4567-2700 (P.D.). Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions.
    [Show full text]