Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2014 Allosteric Activation of Human Glucokinase Joseph M. Bowler Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES ALLOSTERIC ACTIVATION OF HUMAN GLUCOKINASE By JOSEPH M. BOWLER A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Fall Semester, 2014 Joseph M. Bowler defended this dissertation on November 4, 2014. The members of the supervisory committee were: Brian Miller Professor Directing Dissertation Branko Stefanovic University Representative Timothy Logan Committee Member Wei Yang Committee Member Lei Zhu Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements. ii ACKNOWLEDGMENTS I would like to express my deepest gratitude to my advisor and mentor, Dr. Brian Miller. I consider myself fortunate to have had the level of guidance and support in which you have provided throughout my graduate career. Your enthusiasm for discovery will continue to motivate my growth as a scientist. I thank my incredible labmates who have helped me through these years, especially Dr. Mioara Larion. I have such an appreciation for the time you spent training me and your constant willingness to help those around you succeed. To my committee members, Professors Logan, Stefanovic, Yang, and Zhu: thank you for taking the time to provide advice and guidance through meetings and informal interactions. I extend my appreciation to collaborators for providing the opportunity to address topics that might otherwise be inaccessible to a biochemist: Dr. Armen Zakarian, Dr. Mike Roper, Dr. Raghuram Dhumpa, and Dr. Yu Chen. I thank the patient folks who assisted in my experiments or were involved in teaching me proper usage of new instrumentation: Dr. Mark Kearley, Dr. Katherine Hervert, Dr. Lei Zhu, Dr. Claudius Mundoma, and Dr. Umesh Goli. The chemistry department staff members have been exceptionally pleasant and always happy to help with repairs or repurposing of spaces, especially Keith Collins, Mike Fennell, Kevin Kiley, Gary Poplin, Dave Stewart, and Mike Tharp. This work would not exist without my mother and sister. Mom and Melissa, thank you both for encouraging and supporting me for as long as I can remember. Heidi, I cannot express enough gratitude for your presence in my life these past few years. Thank you for all the support and inspiration you've given me. Finally, I want to thank Meagan. You are my motivation to make tomorrow better than today. iii TABLE OF CONTENTS List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abstract ............................................................................................................................................x 1. INTRODUCTION ...................................................................................................................1 1.1 Glucokinase: A Unique Isozyme of the Hexokinase Family .........................................1 1.2 The Physiological Significance of Glucose and the Role of Glucokinase .....................2 1.3 A Monomeric Enzyme with Positive Kinetic Cooperativity .........................................3 1.4 Allosteric Activators of Human Glucokinase ................................................................7 1.5 Observations of Multiple Conformations of Human Glucokinase ................................8 1.6 A Functional Analysis of Glucokinase Activation ......................................................12 1.7 The Future of Glucokinase Activators as Therapeutics ...............................................15 1.8 Aims of the Study ........................................................................................................16 2. INVESTIGATIONS INTO THE MECHANISM OF GLUCOKINASE ACTIVATORS ...18 2.1 Introduction ..................................................................................................................18 2.2 Materials and Methods .................................................................................................22 2.3 Results ..........................................................................................................................28 2.4 Discussion ....................................................................................................................42 3. IN SEARCH OF GLUCOKINASE-ACTIVATING BIOMOLECULES .............................50 3.1 Introduction ..................................................................................................................50 3.2 Materials and Methods .................................................................................................53 3.3 Results ..........................................................................................................................54 3.4 Discussion ....................................................................................................................60 4. USING A PLASMID-BASED CYCLIC PEPTIDE LIBRARY TO GENETICALLY SELECT FOR NOVEL GLUCOKINASE ACTIVATORS .................................................63 4.1 Introduction ..................................................................................................................63 4.2 Materials and Methods .................................................................................................67 4.3 Results ..........................................................................................................................71 4.4 Discussion ....................................................................................................................76 4.5 Future Directions .........................................................................................................79 APPENDICES ...............................................................................................................................83 A. A NOVEL MECHANISM OF GLUCOKINASE ACTIVATION .......................................83 A.1 Introduction ..................................................................................................................83 iv A.2 Materials and Methods .................................................................................................85 A.3 Results ..........................................................................................................................86 A.4 Discussion ....................................................................................................................88 B. SPECTRAL DATA FOR GKA22 SYNTHESIS ..................................................................90 C. MOLECULAR DOCKING CANDIDATES FOR GCK-GKRP DISRUPTORS ...............100 D. SUPPLEMENTARY FIGURES .........................................................................................102 E. THE AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE LICENSE TERMS AND CONDITIONS ...........................................................................104 REFERENCES ............................................................................................................................111 BIOGRAPHICAL SKETCH .......................................................................................................124 v LIST OF TABLES 2.1 Thermodynamic Parameters Obtained via ITC .................................................................33 2.2 Equilibrium Dialysis Assay Results...................................................................................34 2.3 Control Assays for ATP Calculation .................................................................................35 2.4 Transition Time (�) Measurements ....................................................................................39 2.5 Thermodynamic Parameters of Activator Binding ............................................................44 3.1 Fold-Activation Over Control (Rate+EFFECTOR/Rate-EFFECTOR) ...........................................58 3.2 Comparative Measurements of Tatanan Kinetics ..............................................................60 4.1 Comparative Assessment of Cyclic Peptide Libraries .......................................................72 4.2 First Positive Hits From Library-1 Selections ...................................................................78 A.1 Kinetic and Thermodynamic Characterization of GCK Variants ......................................88 C.1 Potential GCK-GKRP Disruptors from Docking Simulations ........................................100 vi LIST OF FIGURES 1.1 The reaction catalyzed by human glucokinase ....................................................................1 1.2 Kinetic saturation plot for GCK showing its delayed response to increasing glucose ........3 1.3 The mnemonical model for positive cooperativity as it applies to glucokinase ..................5 1.4 The ligand-induced slow transition model for cooperativity as it applies to glucokinase ...6 1.5 The first glucokinase activator .............................................................................................7