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Investigations Into Intracellular Thiols of Biological Importance

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Investigations Into Intracellular Thiols of Biological Importance Investigations into Intracellular Thiols of Biological Importance by Christine Elizabeth Hand A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2007 © Christine Elizabeth Hand 2007 AUTHOR'S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The presence of thiols in living systems is critical for the maintenance of cellular redox homeostasis, the maintenance of protein thiol-disulfide ratios and the protection of cells from reactive oxygen species. In addition to the well studied tripeptide glutathione (γ-Glu-Cys-Gly), a number of compounds have been identified that contribute to these essential cellular roles. Many of these molecules are of great clinical interest due to their essential role in the biochemistry of a number of deadly pathogens, as well as their possible role as therapeutic agents in the treatment of a number of diseases. A series of studies were undertaken using theoretical, chemical and biochemical approaches on a selection of thiols, ergothioneine, the ovothiols and mycothiol, to further our understanding of these necessary biological components. Ergothioneine is present at significant physiological levels in humans and other mammals; however, a definitive role for this thiol has yet to be determined. It has been implicated in radical scavenging in vivo and shows promise as a therapeutic agent against disease states caused by oxidative damage. Given the clinical importance of this intracellular thiol, further investigation into the behaviour of ergothioneine appeared warranted. A high level theoretical study was performed to determine the thermodynamic driving force behind the instability of the ergothioneine disulfide, as well as the thermodynamics of the reactions of ergothioneine with a selection of biologically relevant reactive oxygen species. These results were compared to those determined for a glutathione model compound, as well as the related ovothiols. The latter are believed to act as hydrogen peroxide scavengers in vivo and are currently under review as possible therapeutics against oxidative damage. The structural differences between the ovothiols and ergothioneine dramatically affect their reactivity and this study investigates the thermodynamic driving forces behind these differences. Mycothiol is the major thiol found in the Actinomycetales bacteria, which include the causative agent of tuberculosis, and the enzymes which use mycothiol have been identified as important targets for the development of novel antimicrobials. To better understand the in vivo behaviour of mycothiol, a thorough conformational search was performed to determine what, if any, trends exist among the low energy conformers expected to be present in solution. Knowledge of the conformations preferred by mycothiol may aid in the design of substrate-based inhibitors targeted at mycothiol-dependent enzymes. In addition, the efforts towards the identification of a mycothiol-dependent glyoxalase system are described. The glyoxalase system is essential for the detoxification of methylglyoxal, a toxic by-product of glycolysis, and this system would serve as a target for the design of new therapeutics against tuberculosis and other pathogenic Actinomycetales bacteria. In addition to the study of intracellular thiols, this work details a preliminary theoretical study of the thermodynamics of the phosphorylation of proteinaceous serine residues by inositol pyrophosphates in eukaryotic cell-free extracts. It has been postulated that this observed activity may represent a novel signalling pathway in eukaryotes. This study focused on the effect of inositol pyrophosphate structure and overall charge on the thermodynamics of these reactions. This information should contribute to our understanding of this novel cellular phosphorylation process. iii Acknowledgements I am indebted to Dr. John F. Honek for his friendship, guidance and encouragement during the course of this work. This project presented a wide range of challenges and John’s constant patience, faith and enthusiasm was always appreciated. I would like to thank the members of the Honek lab who have been an excellent source of knowledge, laughter and support: Pei Chun Hang, Danish Khan, Meijun Lu, Kadia Mullings, Jennifer Steere, Nicole Sukdeo, Dr. Zhengding Su, Uthaiwan Suttisansanee, Dr. Mark Vaughan, and Paula Walasek. Dr. Elisabeth Daub was delightfully supportive and assisted in all aspects of the microbiology detailed herein. Many thanks to the members of my committee for their valuable advice over the course of my doctorate: Drs. France-Isabelle Auzanneau, Gary Dmitrienko, and Michael Palmer. In addition, I would like to thank my external examiners, Drs. Barbara Moffatt and Jeffrey Atkinson for their participation in this process. The technical assistance of Jan Venne, Robyn Landers, Val Goodfellow and Dr. Richard Smith was greatly appreciated. Dr. Nicholas J. Taylor performed the crystal structure determination of ergothioneine and was very helpful in the preparation of this manuscript. Sadly, Nick passed away before the completion of my degree and I am sure he will be missed by the department. The arduous task of proof-reading this work fell to Emelyne Das, and Drs. Darren Anderson and Jennifer Belelie. I am very grateful for their quick reading, suggestions and support. Luna Guha’s assistance was also appreciated. I have been blessed in having two fantastic mentors: Drs. Janet Kumita and Jennifer Belelie. They have been patient sources of advice, encouragement and unfailing support for which I will always be grateful. I am lucky to have been befriended by many wonderful people during my stay at UW, including Dr. Bryan Hill, Joe and Karen Meissner, Don and Jill Spratt, Jason Yaeck and Andrea Dupont, as well as Kristen Day and Scott Ruttgaizer, who are my Waterloo family. Finally, I must thank Mom, Dad, Brandon, Gramma, Sean, and Heather, as well as Uncle Brian and his family, for all of their advice, encouragement and assistance. Bran.: Thank you for taking such good care of me; your calm perspective and unfailing support was a god- send. Mom and Dad: I can not even begin to express my gratitude; your thoughtfulness, generosity and love overwhelms me. iv For my parents, Bob and Linda Hand “A happy family is but an earlier heaven” ~John Bowring v Table of Contents Abstract ................................................................................................................................ iii Acknowledgements........................................................................................................... iv Dedication..............................................................................................................................v Table of Contents.............................................................................................................. vi List of Tables ...................................................................................................................... xi List of Figures ....................................................................................................................xv Table of Abbreviations................................................................................................ xviii Chapter 1: Introduction to Intracellular Thiols ......................................................1 1.1. Ergothioneine.....................................................................................................................2 1.1.1. Distribution..........................................................................................................2 1.1.2. Biosynthesis.........................................................................................................3 1.1.3. Chemical Properties.............................................................................................3 1.1.4. Biological Function..............................................................................................4 1.2. Ovothiols.............................................................................................................................5 1.2.1. Distribution..........................................................................................................5 1.2.2. Biosynthesis.........................................................................................................6 1.2.3. Chemical Properties.............................................................................................7 1.2.4. Biological Function..............................................................................................7 1.3. Glutathione.........................................................................................................................9 1.3.1. Distribution..........................................................................................................9 1.3.2. Biosynthesis.......................................................................................................10 1.3.3. Chemical Properties...........................................................................................10 1.3.4. Biological Function............................................................................................10
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