Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1994 Expression, purification, characterization, and site- directed mutagenesis of phosphorylase kinase [upsilon] subunit Chi-Ying F. Huang Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons Recommended Citation Huang, Chi-Ying F., "Expression, purification, characterization, and site-directed mutagenesis of phosphorylase kinase [upsilon] subunit " (1994). Retrospective Theses and Dissertations. 10611. https://lib.dr.iastate.edu/rtd/10611 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]. 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Huang A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department : Biochemistry and Biophysics Major: Biochemistry Approved : Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. For the Maj or^epartment Signature was redacted for privacy. For/lft^"^Graduate College Iowa State University Ames, Iowa 1994 ii DEDICATION To My Dearest Wife, MEI-LING. iii TABLE OF CONTENTS ABBREVIATIONS v CHAPTER 1: GENERAL INTRODUCTION 1 General properties of phosphorylase kinase 1 Roles of subunits 4 Inclusion bodies and renatured recombinant proteins 7 Second structural analyses of mutant proteins 9 Substrate specificity 10 Localization of substrate and metal ion binding 14 sites by scanning mutations Kinetic mechanism of recombinant -yi-aoo 21 Specific aims 24 Dissertation organization 25 CHAPTER 2: EXPRESSION, PURIFICATION, CHARACTERIZATION, 27 AND DELETION MUTATIONS OF PHOSPHORYLASE KINASE 7 SUBUNIT: IDENTIFICATION OF AN INHIBITORY DOMAIN IN THE 7 SUBUNIT ABSTRACT 28 INTRODUCTION 29 MATERIALS AND METHODS 32 RESULTS AND DISCUSSION 51 ACKNOWLEDGMENT AND DEDICATION 69 REFERENCES 69 iv CHAPTER 3 : MUTATIONAL ANALYSES OF METAL AND SUBSTRATE 77 BINDING SITES OF PHOSPHORYLASE KINASE y SUBUNIT SUMMARY 79 INTRODUCTION 81 MATERIALS AND METHODS 84 RESULTS 88 DISCUSSION 107 ACKNOWLEDGMENTS 113 REFERENCES 114 CHAPTER 4: IDENTIFICATION OF THE PSEUDOSUBSTRATE 118 BINDING SITE OF PHOSPHORYLASE KINASE 7 SUBUNIT ABSTRACT 119 INTRODUCTION 120 MATERIALS AND METHODS 125 RESULTS 127 DISCUSSION 143 ACKNOWLEDGMENTS 150 REFERENCES 150 CHAPTER 5: GENERAL SUMMARY AND DISCUSSION 154 ADDITIONAL LITERATURE CITED 162 ACKNOWLEDGMENTS 168 V ABBREVIATIONS cAMP 3', 5'-cyclic adenosine monophate cAPK cAMP dependent protein kinase DTT Dithiothreitol EDTA Ethylenediaminetetraacetate EGTA [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High-performance liquid chromatography PhK phosphorylase kinase PIPES Piperazine-N,N'-bis(2-ethanesulfonic acid) SDS Sodium dodecyl sulfate Tris Tris(hydroxymethyl)aminomethane 1 CHAFER 1 GENERAL INTRODUCTION General properties of phosphorylase kinase Protein kinases are defined as the enzymes catalyzing the transfer of the 7-phosphate of MgATP to an acceptor amino acid in a protein substrate. The reaction catalyzed by protein kinases is called phosphorylation. The protein kinases differ in substrate specificity, subunit composition, activation mechanism, but all the eukaryotic protein kinases are found to share sequence similarities throughout a conserved catalytic core (1). The protein kinases are a large family of enzymes, and phosphorylase kinase (PhK) is one of them. PhK has a subunit composition of (aPyô)^ and a molecular weight of 1.3x10® kDa (2) . There are two major pathways for the regulation of glycogenolysis involving PhK. The first pathway is hormonal stimulation of glycogenolysis (3-5). In the presence of epinephrine or glucagon, an increased cyclic AMP triggers the activation of cyclic AMP-dependent protein kinase (cAPK), which activates PhK by phosphorylation on the of and j8 subunit. The activated PhK catalyzes the phosphorylation of glycogen phosphorylase b, the inactive form, and converts it into 2 the active a form which catalyzes the breakdown of glycogen to glucose-l-phosphate. The other cascade is involved in the neural and cx- adrenergic hormonal control of glycogenolysis. These control systems are mediated by Ca^* which serves a mediator to link glycogenolysis to muscle contraction (6- 9). In the resting state of skeletal muscle, the Ca^* concentration in the cytosol is less than 10'® M. Muscle contraction is triggered by the release of Ca^* (10'® to 10'® M) from sarcoplasmic reticulum. Because PhK is highly sensitive to Ca^* concentrations and muscle contraction is also a Ca^* dependent manner, PhK is thought to coordinate the muscle contraction with glycogenolysis. There are two modes of PhK activation by Ca^*. First, Ca^* binds to 6 subunit (calmodulin) of PhK and stimulates its Ca^* dependent activity. Second, exogenous calmodulin binds to the calmodulin binding sites on a and 0 subunits in a Ca^* dependent manner, which stimulates PhK activity. PhK recently has been shown to be a dual specificity kinase (10). The specificity of PhK is dependent on the metal ions; Mg^* activates seryl phosphorylation of phosphorylase Jb and Mn^* causes tyrosyl phosphorylation of angiotensin II. In addition to the known glycogenolysis cascade, new substrate(s) and pathway(s) might exist for 3 PhK to reflect its tyrosine kinase activity. PhK is a very large enzyme and its regulation is very complicated (11, 12). In addition to multi-site phosphorylation, pH, ADP, glycogen, calmodulin, and metal ions, such as Ca^*, Mg^*, Mn^*, affect its activity. PhK activity was shown to be activated by Mg^*. Its activity was activated by Mn^* (MnATP) and then inhibited by increasing Mn^* (or free Mn^*) (13). The location of the metal ion binding sites in y will be described in PAPER 3. A lower kinase activity at pH 6.8 than at 8.2 was found in nonactivated PhK (the activity ratio (pH 6.8/pH 8.2) is 0.04). The pH dependency is changed as the holoenzyme undergoes phosphorylation, proteolysis, or dissociation into ccyô and 7Ô complexes (14). For example, the ratio of activity at pH 6.8/pH 8.2 ranges 0.3-1 for PhK which is phosphorylated by cAPK or by autophosphorylation, respectively. It has been suggested that there are two ADP binding sites per a(3yô tetramer. ADP is known to be an allosteric activator of nonactivated PhK. The allosteric site for activation is in the /3 subunit (15) . ADP is also the product of the kinase reaction and has been shown to be a competitive inhibitor of 7 subunit activity (see later in kinetic mechanism of recombinant 71.300) • This ADP binding site is believed to be in the active site of the 7 subunit 4 (16) Roles of subunits Two isozymes of PhK have been identified and isolated from white (fast-twitch) and red (slow-twitch) muscles. The difference is due to the largest subunits, designated a and a' (17, 18). The rabbit white skeletal muscle PhK composed of four different subunits with a stoichiometry of (a/Syô)^ (19). The molecular weights for a, 13, y, and Ô subunits are 138,422 (20), 125,205 (21), 44,673 (22), and 16,680 (23), respectively. The Ô subunit is calmodulin, a calcium binding protein, and confers calcium sensitivity upon PhK (24). The ô subunit differs from bovine brain calmodulin in only two amino acids and has the ability to activate calmodulin- dependent enzymes (25). Calmodulin acts as a Ca^*- dependent mediator to control a variety of enzymes responding to physiological fluxes of Ca^*. It has been suggested that PhK requires Ca^* for its activity (7) and the binding of Ca^* is through the ô subunit (26, 27). The 7 subunit was shown to contain a catalytic site (28-33) . One of the early experiments was to use lithium bromide to promote the dissociation of PhK. Two partial complexes, Û17Ô and yô, obtained by Chan and Graves, both of 5 which retained the catalytic activity of PhK, indicating the catalytic site is in the yô complex but not in the a and j8 subunit (29, 30). Later, the isolation of active y subunit alone further demonstrated that the y subunit is the catalytic subunit (16, 32).