University of Huddersfield Repository Ali, Antasar Mohamed Biochemical Characterization of a Novel Mammalian Polyphosphate Dependent Glucokinase Original Citation Ali, Antasar Mohamed (2016) Biochemical Characterization of a Novel Mammalian Polyphosphate Dependent Glucokinase. Doctoral thesis, University of Huddersfield. This version is available at http://eprints.hud.ac.uk/id/eprint/32663/ The University Repository is a digital collection of the research output of the University, available on Open Access. Copyright and Moral Rights for the items on this site are retained by the individual author and/or other copyright owners. 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For more information, including our policy and submission procedure, please contact the Repository Team at: [email protected]. http://eprints.hud.ac.uk/ BIOCHEMICAL CHARACTERIZATION OF A NOVEL MAMMALIAN POLYPHOSPHATE DEPENDENT GLUCOKINASE By Antasar Mohamed Ali Supervisor Dr.Shamus Burns A thesis submitted to the University of Huddersfield in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Huddersfield (2016) i Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgment has been made in the text. ii Acknowledgement I am indebted to many people who have helped me throughout the course of this thesis. I have had a huge opportunity to learn and apply my theoretical and practical knowledge in accomplishing this research. First of all I would like to thank my family for giving me inspiration and support all the way through and for patiently helping me to handle the stress in critical stages of this thesis. I would like to pay my heartiest gratitude to my great supervisor Dr Shamus Burns for his continuous support, guidance and advice to complete the research. His directions and cooperation enlightened me to accomplish this thesis to fulfil particular research objectives. Therefore, I’m really indebted to him. I pay heartiest gratitude to my friends and my fellow workmates for giving me inspiration and continuous support in way of completing this thesis. Finally, thanks to everyone whoever, directly and indirectly supported me in completing my thesis. Without their support and contribution, it would be hard to complete my research. iii Abstract Hexokinases are a family of enzymes that catalyse the phosphorylation of glucose by transferring the γ- phosphoryl group from adenosine triphosphate (ATP) to the sixth position hydroxyl group of glucose to generate Glucose 6-phosphate (G6P). Until now, five isozymes of mammalian hexokinase (HK) have been described: types I, II, III and IV, all of them ATP dependent, and type V which is ADP-dependent. The present thesis describes a novel hexokinase, we have designated as PPGKm. The enzyme is strictly polyphosphate (pp) dependent, is correctly defined as a glucokinase (GK), by virtue of its kinetics and is present in mammalian tisues, at high activity in liver. The enzyme does not use ATP and ADP and indeed appears to be inhibited by them. As far as can be ascertained this is the first description of a mammalian enzyme using inorganic polyphosphate as a phosphoryl donor. Polyphosphate is used as phosphoryl donor in bacterial systems and is quite well characterized, although only two micro-organisms have been shown to be strictly pp- dependent like the one described here. This novel enzyme (PPGKm) also showed unique features, differing from the others hexokinases in having a longer half-life and can be stored for several months at -20˚C without loss of activity. However, thermal stability was lower than other hexokinases studied. The enzyme activity is concentrated in the hepatocyte nucleus has a higher molecular weight compared to Hexokinases 1, 2 and 3. The full biological significance of the enzyme is as yet unclear, and attempts to purify and sequence it have been only partly successful. Its specific role in cellular, and especailly nuclear metabolism remains unknown. iv Abbreviations ADP Adenosine diphosphate ANOVA Analysis of variance AP4A Diadenosin tetraphosphate AP5A Diadenosin pentaphosphate ATP Adenosine triphosphate CTP Cyctidine triphosphate DAPI 4',6-diamidino-2-phenylindole Ddppk1 Eukaryotes homolog to bacterial polyphosphate kinase1. DH2O Distilled water D2O Deuterium oxide DTE 1, 4-Dithioerythritol DTT Dithiothreitol ESI-MS Electrospray ionization mass-spectroscopy F1P Fructose 1 phosphate F6P Fructose 6 phosphate FGF Fibroblast growth factor FK Fructokinase G1P Glucose 1phosphate G6P Glucose 6 phosphate GK Glucokinase GKRP Glucokinase regulatory protein GTP Guanosin triphosphate HK Hexokinase HKDC1 Hexokinase domain containing 1 HMP Hexamethaphosphate HPLC High pressure liquid chromatography HSP70 Heat shock protein70 LC-MS/MS Liquid chromatography mass-spectroscopy MCF7 Human carcinoma cell line mTOR Mammalian target of rapamycine MODY2 Maturity onset diabetes of the young type2 MS Masss-pectroscopy NAD Nicotinamine adenine dinucleotide (oxidized form) NADH Nicotinamine adenine dinucleotide (reduced form) NDK Nucleotide diphosphate kinase Na HEPES Sodium salt (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) NMR Nuclear magnetic resonance PAP Polyphosphate: AMP phosphotransferase PCs Human plasma cells Pi Inorganic phosphate v P700 Polyphosphate of 700 chain length PBS Phosphate buffer saline PHB Polyhydroxybutyrate Poly-Pi60 Polyphosphate has chain length of 60 phosphates PPGK Polyphosphate glucokinase PPGKm Mammalian polyphosphate glucokinase Poly-Pi Polyphosphate PPK1 Polyphosphate kinase 1 PPX Exoployphosphatase Pvds RNA polymerase Sigma factor gene of Pseudomonas aeruginosa . RecA Gene encodes DNA-recombinase Rpos Gene which encodes RNA polymerase sigma factor SDS-PAGE Sodium dodecyl sulphate- polyacrylamide gel electrophoresis SOS Global response to DNA damage TOR Target of rapamycine Tri-P Tripoly phosphate Tris (hydroxymethyl) aminomethane TSP Trimethlsilyl propanoic acid UTP Uridine triphosphate VDAC Voltage -dependent anion channel vi Units: µg Microgram mg Milligram mM Millimolar mL Milliliter ˚C Degree celsius V/V Volume/Volume W/V Weight /Volume IU International unit Ppm Part per million vii Table of Contents Declaration ........................................................................................................................................... ii Acknowledgement ............................................................................................................................... iii Abstract ................................................................................................................................................iv Abbreviations ........................................................................................................................................ v Table of Contents .............................................................................................................................. viii List of Figures..................................................................................................................................... xii List of Tables ......................................................................................................................................xvi Chapter 1 Introduction ........................................................................................................................... 1 Part One: Evolution of Hexokinases Isoenzymes and their regulatory role ................................ 1 1.1.1 Sugar specificity and naming conventions ................................................................................... 2 1.1.2 Molecular mass of hexokinases in different phyla ....................................................................... 4 1.1.3 Tissue distribution of hexokinase isoenzymes in mammalian systems ........................................ 5 1.1.4 Subcellular Distribution of Isoenzymes ....................................................................................... 7 Part Two: Introduction to polyphosphate .................................................................................. 20 1.2.1 The main functions of polyphosphate in cells of living organisms ............................................ 21 1.2.2 Intracellular polyphosphate levels and subcellular distribution ................................................. 29 1.2.3 Main enzymes of polyphosphate metabolism............................................................................. 32 1.2.4 Applications of polyphosphate ................................................................................................... 43 1.2.5 Various forms of Polyphosphate in cells ...................................................................................