Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1999 The glycogen phosphorylase/ phosphorylase kinase interaction: effects of mutations in the amino-terminal region of glycogen phosphorylase Alyssa Christine Biorn Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Biorn, Alyssa Christine, "The glycogen phosphorylase/ phosphorylase kinase interaction: effects of mutations in the amino-terminal region of glycogen phosphorylase " (1999). Retrospective Theses and Dissertations. 12198. https://lib.dr.iastate.edu/rtd/12198 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Belt & Howell Information and Learning 300 North Zeeb Road, Ann Artxsr, Ml 48106-1346 USA 800-521-0600 The glycogen phosphorylase/ phosphorylase kinase interaction: Effects of mutations in the araino-terminal region of glycogen phosphorylase by Alyssa Christine Biom A dissertation submitted to the graduate facility in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Molecular, Cellular and Developmental Biology Major Professor: Donald J. Graves Iowa State University Ames, Iowa 1999 UMI Nxjmber: 9941789 UMI Microform 9941789 Copyriglit 1999, by UMI Company. All rights reserved. This microform edition is protected agaiast unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ii Graduate College Iowa State University This is to certify that the Doctoral dissertation of Alyssa Christine Biom has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Signature was redacted for privacy. Signature was redacted for privacy. iii TABLE OF CONTENTS GENERAL INTRODUCTION 1 Glycogen Phosphorylase 1 Early history of Glycogen Phosphorylase 1 General GP characteristics 4 Regulation of GP activity 8 Conformation of phosphorylase 9 Phosphorylase Kinase 12 General characteristics 12 The Y catalytic subunit 13 Substrate specificity 15 Interaction of Phosphorylase with Phosphorylase Kinase 17 Experimental Rationale and Dissertation Organization 19 References 20 SITE-DIRECTED MUTANTS OF GLYCOGEN PHOSPHORYLASE ARE ALTERED IN THEIR INTERACTION WITH PHOSPHORYLASE KINASE 26 Abstract 26 Introduction 27 Experimental Procedures 29 Materials 29 Mutagenesis 29 Protein Expression and Purification 30 Phosphorylation Assays 30 Kinetics 31 Results 32 Mutagenesis 32 Interaction of mutants with Y(1-300) 33 Interaction of GP mutants with a mutant of Y(1-300), yEllOK 34 Interaction of GP mutants with Protein ECinase A 35 Discussion 36 References 41 GLYCOGEN PHOSPHORYLASE LIGAND-BINDING PROPERTIES ARE ALTERED BY SINGLE SITE MUTATIONS IN ITS AMINO- TERMINAL REGION 60 Abstract 60 Introduction 61 iv Experimental Procedures 63 Materials 63 Mutagenesis 64 Protein Expression and Purification 64 AMP Activation Assays 65 Phosphorylation Assays 65 Results 66 Activity and AMP-binding characteristics 66 Conformational effects on phosphorylation 68 Phos. a activity of GP mutants 69 Discussion 70 References 76 GENERAL CONCLUSIONS 97 ACKNOWLEDGMENTS 101 1 GENERAL INTRODUCTION Glycogen Phosphorylase Early history of Glycogen Phosphorylase Muscle glycogen phosphorylase (a-l,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) is an important en2yme in carbohydrate metabolism. The homodimeric enzyme utilizes inorganic phosphate in the removal of one glucose unit from the non-reducing end of the storage polysaccharide, glycogen. a-D-Glucose-l-phosphate is produced in this reaction, which is then converted to glucose-6-phosphate, used for the formation of ATP, the cell's energy soxirce. Glycogen phosphorylase (GP) is one of the longest- and most extensively-studied enzymes and has a rich and interesting "history". Glycogen phosphorylase was first discovered as a phosphorylase activity towards glycogen in rabbit skeletal muscle extracts by Carl and Gerty Cori in the mid-1930's (1). This phosphorylase activity required adenosine-5'- monophosphate (AMP) for activity and was shown to release the product glucose-l-phosphate (GIP) (2). The AMP required for activity was thought to be tightly bound to the phosphorylase protein and to be catalytic; in other words, a coenzyme. In time, two forms of phosphorylase were shown to exist: one which had no activity in the absence of AMP, termed phosphorylase ^ and one form which had 60-70% of its maximal activity in the absence of added AMP, termed phosphorylase a (3). Because phos. b required AMP for activity and phos. a did not, it was assumed that AMP was already present in the phos. a form, and therefore did not require its addition (3). During the isolation of phosphorylase, another protein was found in the muscle extracts which converted phos. a to phos. b. The enzyme was named the 2 "PR Enzyme" for "prosthetic removing", as it was assumed to remove the prosthetic group AMP from phos. a (4), even though no free adenylic add could be detected after incubation of phos. a with PR. Keller and Cori, in 1953, proposed a change of the name from "prosthetic-removing" to "phosphorylase- rupttiring" when they foimd that, along with the conversion of phos. a to b, PR caused a halving of the weight of phosphorylase, as determined by sedimentation velocity (5). They could not say how this halving was accomplished, however. At about the same time another group of investigators, Edwin Krebs and Edmond Fischer, found mostly phos. b when they purified muscle phosphorylase, while the Cori group obtained phos. a (6). They determined that this difference was not due to contaminating PR enzyme in the phosphorylase preparations. The only difference between their methods was that Krebs and Fischer did not filter the extracts through paper. Until this time, phos. a was believed to be the GP form found in resting muscle, but this work of BCrebs and Fischer showed that it was phos. b instead. They found that the presence of contaminating divalent metal ions either in tap water used for extraction, or on the filter papers, caused conversion of phos. b to phos. a during the purification process (7). They could also cause a conversion of phos. b to a by adding crude muscle extracts plus divalent metal ioris to purified phosphorylase b. ATP was needed, however, if the extract was not used right away. In addition, this conversion was inhibited by EDTA. Krebs and Fischer believed this conversion process to be enzyme-catalyzed, as a protein fraction of the extract was also required (7). In 1956, this protein fraction was isolated and studied further as the phosphorylase "converting enzyme" (8). Using ^^P, they demonstrated that 2 3 mol were incorporated into 1 mol of phosphorylase b upon incubation with converting enzyme. This was the first demonstration of phosphate incorporation into GP and led Krebs and Fischer to believe that this might be a kinase reaction (8). Kinases had not previously been observed to have protein substrates. Importantly, they also fotmd that 32p was released when 32p-phos- a was converted to phos. b by the PR enzyme (8, 9). The work of all these researchers was extremely significant in that this was the first observation of reversible phosphorylation of an enzyme to regulate its activity. We now know the "converting enzyme" to be phosphorylase kinase (10), and "PR" to be phosphorylase phosphatase (II), or protein phosphatase-1 (12). These two enzjnnes work in concert to modulate the activity of phosphorylase to control the rate of glycogenolysis. This work made up the first twenty years of study on glycogen phosphorylase and its complex system of regulation. It is very interesting to read these older papers and discover how insightful the researchers of the time were about such a new kind of enzymatic regulation. Glycogen