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24666Hakda.Pdf University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies Legacy Theses 1997 The properties of beta-galactosidases from Escherichia coli with substitutions for glycine 794 and tryptophan 999 Hakda, Shamina Hakda, S. (1997). The properties of beta-galactosidases from Escherichia coli with substitutions for glycine 794 and tryptophan 999 (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/13125 http://hdl.handle.net/1880/26631 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca THE UNZVERSITY OF CALGARY The hperties of 8-Galactosidases hmEscherichia coli With Substitutions for Glycine 794 and Tryptophan 999 Shamina Hakda A THESIS SUBMlTTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY,ALBERTA AUGUST, 1997 O Shamina Hakda 1997 National Library Bibliothèque nationale of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON KiA ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distrhute or seil reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/Eilm, de reproduction sur papier ou sur format électronique. The author retains ownershp of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantieIs may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Substitution of Ala for Gly-794 is thought to lock the loop made up of residues 793 to 804 near to the active site. This substituted enzyme caused poor binding of hydrophobic groups ta the glucose site compared to the wild type enzyme. Some transition state analog inhibitors bound better to this substituted enzyme. Some galactosylation rates were also better. In addition, glucose bound to the free form of the substituted enzyme much better than to the free form of the wild type enqnne, The locking of the position of the loop seems to change the conformation of the enzyme fiom the free form to the conformation of the enzyme after the glycosidic bond is cleaved. Glucose also bound much better to the galactosyl form of the enzyme but reacted more poorly to form allolactose. Substitution of Phe or Gly for Trp-999 in the aglycone site or glucose subsite of B-galactosidase caused dramatic decreases of the hydrop hobicity of this glucose subsite. In addition D-glucose bound much more poorly in both the fkee form and the galactosyl form. This is probably due to the loss of the hydrophobic stacking interactions that Trp-999 provides for the hydrophobic side of glucose. The reaction to form allolactose was rapid, but poor binding at the aglycone subsite resulted in low allolactose production. In some cases the galactosylation rate with PNPG was increased 1 would like to sincerely thank my supervisor, Dr. RE. Huber for his kindness, advice, guidance, insight, patience and generosity throughout the years working in his laboratory. His encouragement and enthusiasm made working under his supemision a rewarding learning experience. His evaluation of this thesis was greatly appreciated. 1 would also like to express my gratitude ta a couple of people who made direct contributions to this thesis: Tien Phan for her help with the purification and partial kinetic analysis of W999F-B-galactosidase; and Mark Britton for the purification and partial kinetic anal+ of W999G-B-galactosidase. 1 also thRnk Jasmine Ahmed, Heather Seidle and Beatrice Rob and for their discussions, suggestions, friendship and support. 1 would also like to express my gratitude to Dr. KJ. Stevenson for his kindness, support and generosity. Finally, 1 would like to thank my family for their continuous love, support and encouragement. Dedicated to my parents Approval page i .* Abstract xx Acknowledgments iv Dedication v Table of Contents vi List of Tables xii. List of Figures xv List of Abbreviations and Symbols xi3 1. INTRODUCTION ...................................................................................................... 1 1.1 Glycosidases................................................................................................. 1 1.1.1 Mechanism of Action for Retaining Glycosidases............... -2 1.2 B-Galactosidase: a Brief Description............... .. ...................................4 1.2.1 Reactions Catdyzed by &galactoçidase.. .............................. -5 1.2.1.1 Hydrolytic and Thmgalactosylic Reactions with F Lactose... ................................................................................... .*.u 1.2.1.2 Hydmlytic and Trançgalactosylic Reactions with Synthetic Substrates......,............ .... ..... L 1.2.1.3 ReversionReactions .....................................................9 12.2 Binding Sites ................................................................................ 10 1.26.1 The Galactose Subsite ........................... t... ............... 10 1.2.2.2 The Glucose Subsite ................................................... 13 1.2.3 Reaction Mechanism of l3-Galactosidase.............................. 15 1.2.3.1 General Description .................... ....... .................... 15 1.2.3.2 Evidence for a Two Step Mechankm...................... 15 1.2.3.3 Reaction Pathwa~r:Evidence for a Common Intmmedkte............................................................................... 16 1.2.3.4 Evidence for a Covalent Galactosyl Enzyme Intermediate............................................................................... 17 1.2.3.5 Nature of the Transition State................................. 18 1.2.3.6 The Distinction Between Transition States and Covalent Enzyme htennediates.......................... d 1.2.4 Mg2+ Requirement of Malactosidase........ .... .................... 20 1.2.5 The pHProfile of eGalactosidase........................................... 22 1.2.6 The Structure of eGalactosidase...... .. ................................... 24 1.3 Robing the Active Site of 8-Galactosidase......................................... 26 13.1 Inhibitor Studies....................................................................... -26 1.3.1.1 Determination of Ki and Z(iW...................................... 28 1-32 Site Dù.ected Mutagenesis ....................................................... 28 1.3.2.1 Reaction Profiles......................................................... 30 1.4 Active Site Groups of &Galadosidase......... ,. ................................... 32 1.4.1 Active Site Histidine Residues: His.357, His.391, His.540, His-450 and His-418............................................................................. 32 1.4.2 Glu416......................................................................................... 36 1.4.3 Glu-537......................................................................................... 37 1.4.4 Met-502........................................................................................ 38 1.4.5 Tyr-503 ......................................................................................... 38 1.4.6 Glu-461......................................................................................... 39 1.4.6.1 Glu-461 as an AQd Base Catalyst.......................... 39 1.4.6.2 Rde of Glu461 in Mg2+ Binding............................... 40 1.4.6.3 Role of Glu-461in Transition State vii . Stabiùzation.......................titi. -... ............................................... 41 1.4.6.4 NucleophiIic Activation of B-Galactosidases with Substitutions for Glu-46 ï. ....................................................... 42 1.4.7 Trp-999.............................. ,.. 1.4.8 Gly.79 4. ........................................................................................ 44 1.4.8.1 Gly-794 and hopMovement ....................................4t 2. O~CTIVES............................................................................................... .. ....48 2.1 Gly-794........................................................................................................ -4t 22 Trp-999........................................ .. ........................................................... 4a 3. MATERIALS.......................................................................................................... 50 3.1 Biochemical Reagents ......................,. ..................................................... 5( 3.2 Plasmi&....................................................................................................... 51 3.3 Oligonucleotide Rimers........................................................................... 31- 3.3.1 SequencingPrirner....................................................................
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