EXPLORATION OF THE INTERPLAY BETWEEN ALLOSTERIC INTERACTIONS OF PHOSPHOFRUCTOKINASE FROM RAT LIVER A Dissertation by DAVID ALAN HOLLAND III Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Gregory Reinhart Committee Members, Ry Young Hays Rye Coran Watanabe Head of Department, Gregory Reinhart May 2018 Major Subject: Biochemistry Copyright 2018 David Alan Holland III ABSTRACT The key metabolic enzyme phosphofructokinase (PFK) catalyzes the phosphorylation of fructose-6-phosphate (Fru-6-P) by MgATP in the first committed step of glycolysis. PFK’s phosphorylation activity is tightly regulated by allosteric effectors who act by either increasing or decreasing its affinity to substrate Fru-6-P. Liver PFK from rats (RLPFK) is allosterically regulated by several metabolic byproducts including MgATP, citrate and AMP. Despite the importance of precise regulation of this enzyme, the interplay between the different allosteric effectors has not been thoroughly characterized. Presented here are the effects of MgATP on the ability of allosteric activator (AMP) or inhibitor (citrate) to modulate the activity of RLPFK. We see that MgATP dramatically decreases the ability of both AMP and citrate to allosterically regulate RLPFK. RLPFK has additionally been implemented to be subjected to activation through a novel self-association mechanism. This is difficult to prove unequivocally as the kinetic assays used to measure enzyme activity are performed (by necessity) at concentrations thought to be too low for self-association to occur. It has been demonstrated that at a physiological enzyme concentration Fru-6-P promotes self- association. We propose that if Fru-6-P promotes the formation self-associated species, then self-association must increase its affinity for Fru-6-P. Utilizing Fluorescence Correlation Spectroscopy (FCS) we measured the size of RLPFK particles at concentrations that span the gap between those used for kinetic assays ii and those representing physiological conditions. Additionally, we varied the concentration of ligand (either MgATP or Fru-6-P). Our data demonstrate that Fru- 6-P acts cooperatively with concentration of RLPFK to increase the extent of self- association, whereas no self-association occurs in the presence of MgATP. These FCS data provide exciting insight into the role of activation by self-association and provides the foundation for which future experiments can expound. iii DEDICATION To Kelvin and Alan. May our children’s future be forever greater than our father’s past. iv ACKNOWLEDGEMENTS I would like to thank my boss and committee chair, Dr. Reinhart, for all the mentorship, advice and support that he has provided through the years. I would also like to thank my committee members, Dr. Young, Dr. Watanabe and Dr. Rye for their guidance and support throughout the course of this research. I am grateful for the abundance of technical expertise and scientific advice on fluorescence experiments provided by Dr. Lasagna. In addition, I am grateful for the FCS instrumental and theoretical support that I received through countless phone calls to Dr. Liao. I would like to thank all of the former and current members of the Reinhart lab who provided scientific conversation on a daily basis. Pepe’s has earned a permanent place in my heart and burned a permanent hole in my stomach. Thanks to Dr. Amy Whitaker for endless conversation, scientific and otherwise. You have greatly enriched my time here. Thanks also to friends and colleagues, my softball teams BuckyBallers and Wild Things, the BGA and the entire Biochemistry and Biophysics faculty and staff for making my time at Texas A&M University a great experience. I would like to thank my parents for challenging and encouraging me to be the best that I can be. I would especially like to thank my wife, Michal, for the love and support that she has shown me during this journey. Thank you for putting up with long nights and lonely weekends. Thank you for comforting me when I was v stressed and frustrated, and you only wanted me home. And for sharing in my excitement when I was ecstatic about results that you would otherwise care less about. There really is no way to overstate how important you have been to me completing this thesis, this achievement is as much yours as mine. vi NOMENCLATURE A Substrate ACF Autocorrelation function ATP Adenosine triphosphate ADP Adenosine diphosphate AMP Adenosine monophosphate APD Avalanche photodiode cAMP Cyclic Adenosine monophosphate CPS Counts per second Da Daltons DTT Dithiothreitol E Enzyme EGFP Green fluorescence protein FCS Fluorescence Correlation Spectroscopy FITC Fluorescence Isothiocyanate FRET Forester resonance energy transfer EAM Ensemble allosteric mode EDTA Ethylenediamine tetraacetic acid FPLC Fast protein liquid chromatography Fru-2,6-BP Fructose-2,6-bisphosphate vii Fru-6-P Fructose-6-phosphate ∆Gay Coupling free energy for the binding of substrate and effector Kcat Turnover number Ka Apparent dissociation constant for substrate A Kia0 Dissociation constant for A in the absence of effector Kia∞ Dissociation constant for A in the presence of effector Kiy0 Dissociation constant for Y in the absence of substrate Kiy∞ Dissociation constant for Y in the presence of substrate Km Michaelis constant IPTG Isopropyl β-D-1-thiogalactopyranoside KNF Sequential model LB Lysogeny broth MgATP Magnesium Adenosine triphosphate MOPS 3-(N-Morpholinio)propanesulfonic acid MWC Concerted model NADH Nicotinamide adenine dinucleotide, reduced form NBD Nucleotide binding domain 푛퐻 Hill number Pi Phosphate PEP Phospho-(enol)pyruvate PEG Polyethylene glycol viii PCH Photon counting histogram PFK phosphofructokinase PMSF Phenylmethaneslfonylfluoride PpPFK Phosphofructokinase from Pichia pastoris PSF Point Spread Function Qaa Coupling constant for the binding of two substrates (K-type) Qay Coupling constant for the binding of the substrate and effector (Y) (K-type) Qax Coupling constant for the binding of the substrate and effector (X) (K-type) Qxy Coupling constant for the binding of the effector (X) and effector (Y) (K-type) Qaxy Coupling constant for the binding of substrate and effectors X and Y (K-type) RLPFK Phosphofructokinase from Rat Liver rRLPFK Recombinantly expressed phosphofructokinase from Rat Liver RLPFK-FITC Rat liver phosphofructokinase labeled with fluorescein isothiocyanate SDS-PAGE Sodium sodecyl sulfate polyacrylamide gel electrophoresis SBD Substrate Binding Domain ix 푣 Initial velocity 푉 Maximum velocity X Primary Effector Y Secondary Effector x CONTRIBUTIONS AND FUNDING SOURCES This work was supervised by a dissertation committee consisting of Professor Gregory Reinhart, Professor Ry Young, and Professor Hays Rye of the Department of Biochemistry and Biophysics and Professor Coran Watanabe of the Department of Chemistry. The electron microscopy images presented in Chapter 4 and Chapter 5 were generated by Jeng-Yih Chang in the laboratory of Professor Junjie Zhang from the Department of Biochemistry and Biophysics. All other work for the dissertation was completed independently by the student. This work was made possible in part by the National Institutes of Health under grant number R01 GM 033216, the National Institutes of Health Chemistry- Biology Interface Predoctoral Training Program under grant number T32 GM008523-13A2, the Welch Foundation under grant A-1543, the Willie May Harris Fellowship from Texas A&M University, and support from Texas A&M AgriLife Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the supporting agencies. xi TABLE OF CONTENTS Page ABSTRACT ........................................................................................................................................... ii DEDICATION ....................................................................................................................................... iv ACKNOWLEDGEMENTS ................................................................................................................. v NOMENCLATURE ............................................................................................................................ vii CONTRIBUTIONS AND FUNDING SOURCES........................................................................... xi TABLE OF CONTENTS .................................................................................................................... xii LIST OF FIGURES .............................................................................................................................xiv LIST OF TABLES ............................................................................................................................... xxi CHAPTER I INTRODUCTION ..................................................................................................................... 1 A Brief History of the Theory and Application of Allostery................... 1 Brief History of Phosphofructokinase .......................................................... 31 Fluorescence Correlation Spectroscopy ...................................................... 49 II EXPRESSION, PURIFICATION AND CHARACTERIZATION OF RECOMBINANT RAT LIVER PHOSPHOFRUCTOKINASE .................................................. 60 Introduction .........................................................................................................