DOCSLIB.ORG

ALLOSTERIC REGULATION of PROTHROMBIN ACTIVATION by FACTOR Va

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
ALLOSTERIC REGULATION of PROTHROMBIN ACTIVATION by FACTOR Va ALLOSTERIC REGULATION OF PROTHROMBIN ACTIVATION BY FACTOR Va MAHESHEEMA ALI Bachelor of Science in Botany, Microbiology, Chemistry Osmania University, India April 2000 Master of Science in Organic Chemistry Osmania University, India April 2002 Submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICALCHEMISTRY at the CLEVELAND STATE UNIVERITY May 2016 We hereby approve this dissertation for Mahesheema Ali Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry Degree for the Department of Chemistry and the CLEVELAND STATE UNIVERSITY College of Graduate Studies ________________________________________ Dissertation Chairperson, Dr. Michael Kalafatis Department of CHEMISTRY ______________________ Date ________________________________________ Dissertation Committee Member, Dr. Edward F. Plow Department of MOLECULAR CARDIOLOGY Cleveland Clinic Foundation ______________________ Date ________________________________________ Dr. Anton A. Komar Department of BIOLOGY ______________________ Date ________________________________________ Dr. David J. Anderson Department of CHEMISTRY ______________________ Date ________________________________________ Dr. Crystal M. Weyman Department of BIOLOGY ______________________ Date Date of Defense: April 22nd, 2016 DEDICATION I dedicate this thesis to my loving family. My beloved husband, Mir Ali, has shown unwavering support and encouragement during past five years of my doctoral journey. I am grateful for his love and continuous support, which gave me the strength and courage to pursue my dream and to make it come true. I would like to give special thanks to my wonderful children--Maaz, Maryum, and Idris--for their support and patience throughout. I would like to thank my parents, Mrs. Kaneez Fathima and Mr.Yousuf Ali for all that I have become today, for the constant support in my academic career and personal life. They believed in me and encouraged me to strive in my life. I would like to thank my mother-in-law, Mrs.Moida Bano, and father-in-law Mr. Masood Ali, for all the encouragement of our family. Special thanks also goes to my sisters Mahjabeen, Afreen Fathima, Arshia Fathima and my brothers Kamran Mahmood and Rizwan Mahmood. They were always there when I needed them. The love of family is life’s greatest blessing. I love you all very much. ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation to my advisor, Dr. Michael Kalafatis, a great educator and a wonderful mentor. His invaluable advice and insightful comments have shaped my career for the future. He is patient and kind, and I thank him for the rewarding experience in his research lab. I wish to thank the members of my dissertation committee for their valuable time, collegial guidance and support to help improve my research. I thank Dr. Edward F. Plow, Dr. David J. Anderson, Dr. Crystal M. Weyman, and Dr. Anton A. Komar. I would like to thank Dr. Mary McDonald for reading this text for grammatical and syntactical issues. I especially would like to thank, the department secretaries, Richelle Emery and Michele Jones, for all their administrative help to make this achievement possible. I would like to thank Dr. Jamila Hirbawi for being a wonderful mentor and friend throughout and providing me with great support to be able to accomplish my work. I would like to thank Dr. Joseph Wiencek for his tremendous support. I appreciate his help and support. I would like to greatly acknowledge, Gregory M. Guzzo, research technologist, who has helped me tremendously in my day-to-day activities. I thank you Greg, for being there for me and supporting me as a wonderful friend. I also wish to acknowledge, Katie Turner and Jasmine Manouchehri for being wonderful friends and supporting me. They will never know how much their support meant to me. I would like to thank all my colleagues from Cleveland State University, especially those who have helped me along the way. I would like to thank the entire faculty who have taught and mentored me from Chemistry Department at Cleveland State University. Finally, I thank one and all. Thank you. ALLOSTERIC REGULATION OF PROTHROMBIN ACTIVATION BY FACTOR Va MAHESHEEMA ALI ABSTRACT Upon vascular injury, the proteolytic conversion of prothrombin to thrombin occurs in the presence of prothrombinase. Prothrombinase is an enzymatic complex between factor Va (fVa) and factor Xa (fXa) assembled on a membrane surface in the presence of divalent metal ions. Blood coagulation factor V (FV) is a multi-domain protein (A1-A2-B-A3-C1-C2) with no procoagulant activity and circulates in blood as a procofactor. Our investigation demonstrates that fV is activated by thrombin in a kinetically preferred order, by a first cleavage at Arg709, followed by cleavage at Arg1545 to ultimately generate the active cofactor species, fVa. The cofactor binds to membrane surfaces and effectively serves as receptor for membrane-bound fXa. Although membrane-bound fXa alone is capable of activating prothrombin through initial cleavage at Arg271, followed by the cleavage at Arg320 (Prethrombin-2 Pathway); the rate of activation is not physiologically compatible with survival. However, incorporation of fVa into prothrombinase results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation with the order of cleavages reversed, with initial cleavage at Arg320 followed by Arg271 (Meizothrombin Pathway), resulting in physiologically relevant rates of thrombin formation at the place of vascular injury. We have shown that fXa interacts with prothrombin through amino acid region 478-482 in a fVa-dependent manner. We further demonstrate that basic amino acids from exosite I of prothrombin are involved in recognizing fXa within prothrombinase also in a fVa-dependent manner. Finally, we characterized the allosteric vi interactions of these amino acid residues within prothrombin required for optimum prothrombin activation rates and optimal thrombin function. Our data suggest that amino acids Leu480 and Gln481 allosterically interact with basic amino acids from exosite I on prothrombin, thus modulating the enzymatic activity of fXa within prothrombinase. Our results also provide a logical explanation for the deleterious physiological consequences of natural mutations in proexosite I (Arg382→Cys) and (Arg388→Hys) and demonstrate that patients harboring these mutations are impaired in their ability to form a fibrin clot and, thus, are prone to severe bleeding. Overall our data underline the crucial physiological importance of fVa for thrombin generation and clot formation. vii TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………...vi LIST OF TABLES…………………………………………………………………………….....xi LIST OF FIGURES………………………………………………………………......................xii CHAPTER I INTRODUCTION………………………………………………………………….1 1.1 Cardiovascular Disease and Associated Risk………………………………………1 1.2 Hemostasis…………………………………………………………………………2 1.3 Primary Hemostasis………………………………………………………………..3 1.4 The Coagulation Cascade: Secondary Hemostasis………………………………...4 1.5 Blood coagulation fV……………………………………………………………....9 1.6 Activation of fV………………………………………………………………….11 1.7 fV Inactivation…………………………………………………………………...13 1.8 Formation of the Prothrombinase Complex……………………………………...13 1.9 Interaction of fVa with Phospholipid Membranes……………………………….16 1.10 Interaction of fVa with fXa………………………………………………………18 1.11 Interaction of fVa with Prothrombin……………………………………………..18 1.12 Thrombin…………………………………………………………………………18 1.13 Clinical Significance……………………………………………………………..20 1.14 References………………………………………………………………………..22 CHAPTER II THE MOLECULAR MECHANISM CORRELATING THROMBIN CLEAVAGE AND ACTIVATION OF FACTOR V……………………………………………33 viii 2.1 Abstract………………………………………………………………………...33 2.2 Introduction…………………………………………………………………….35 2.3 Experimental Procedures……………………………………………………....37 2.4 Results…………………………………………………………………………44 2.5 Discussion……………………………………………………………………..62 2.6 References……………………………………………………………………..65 CHAPTER III ALLOSTERIC INTERACTIONS REGULATING PROTHROMBIN ACTIVATION AND THROMBIN ACTIVITY………………………………………………..71 3.1 Abstract………………………………………………………………………71 3.2 Introduction………………………………………………………………….73 3.3 Experimental Procedures…………………………………………………….76 3.4 Results………………………………………………………………………..84 3.5 Discussion…………………………………………………………………...110 3.6 References……………………………………………………………………114 CHAPTER IV OVERALL CONCLUSION……………………………………………………122 ix 4.1 Conclusion……………………………………………………………….122 4.2 References………………………………………………………………126 x LIST OF TABLES Table 2.1 Functional Properties of Recombinant fV Molecules……………………………………….57 Table 3.1 Molecular Weight of Prothrombin Fragments ………………………………………………91 Table 3.2 Kinetic Constants of Wild-Type and Selected rIIa Mutant Molecules toward S-2238…………………………………………………………………………………………..101 Table 3.3 Kinetic Constants of Plasma FII and rFII Mutant Molecules Activation by Prothrombinase…………………………………………………………………………………103 xi LIST OF FIGURES Figure 1.1 The Coagulation Cascade……………………………………………………………...8 Figure 1.2 Schematic of fV………………………………………………………………………10 Figure 1.3 Schematic of Human fV……………………………………………………………...12 Figure 1.4 Prothrombin Activation………………………………………………………………15 Figure 1.5 Prothrombinase Complex…………………………………………………………….17 Figure 2.1 Recombinant Mutants Generated to Explicate the Role of Individual Cleavage Site..……………………………………………………………………………………………...46 Figure 2.2 SDS-PAGE Analysis of Purified Recombinant Proteins (3µg/lane) before (0 min) and after Activation with Thrombin
Load more

Recommended publications
  • Allosteric Regulation in Drug Design
    Mini Review Curr Trends Biomedical Eng & Biosci Volume 4 Issue 1 - May 2017 Copyright © All rights are reserved by Ashfaq Ur Rehman DOI: 10.19080/CTBEB.2017.04.5555630 Allosteric regulation in drug design Ashfaq Ur Rehman1,2*, Shah Saud3, Nasir Ahmad4, Abdul Wadood2 and R Hamid5 1State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, China 2Department of Biochemistry, Abdul Wali Khan University Mardan, Pakistan 3Laboratory of Analytical Biochemistry and Bio separation, Shanghai Jiao Tong University, China 4Department of Chemistry, Islama College University Peshawar, Pakistan 5Department of Bioinformatics, Muhammad Ali Jinnah University Islamabad, Pakistan Submission: May 02, 2017; Published: May 23, 2017 *Corresponding author: Ashfaq Ur Rehman, State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China, Tel: ; Fax: 86-21-34204348; Email: Abstract mechanism, which are initiated through attachment of ligand or inhibitors with the protein or enzymes other than active (orthosteric) sites. ThisProtein mini review and enzymes involved play mechanism, significant types roles and in importancebiological processes of allosteric of all regulations living organisms; in drug theirdesign functions process. are regulated through allosteric Keywords: Allosteric, Activator: Drug design Introduction and ultimately cause disease. While various biological processes expressed the control at different points in life time of protein function is pivotal. As all the cell processes are under carful For the survival of all organisms the significance of protein included regulation of gene expression, translation into protein control and if not properly controls this leads to the abnormality through control of activity and at last degradation of protein [1].
    [Show full text]
  • Enzyme Regulation What Factors Influence Enzymatic Activity?
    12/3/13 Enzyme Regulation What Factors Influence Enzymatic Activity? Principle means of regulating enzyme activity • Reversible, non-covalent (allosteric and simple- MM) – typically small molecules • Reversible, covalent • Protein-Protein interactions • Zymogen activation • Protein expression and degradation • Availability (both of enzyme and substrate) Reversible Noncovalent: Reversible Noncovalent Allosteric Simple activation and inhibition by small molecules – Action at "another site" substrate, natural regulators of enzymes Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors MM kinetics Km, Vmax – competitive, non These effectors are usually produced elsewhere in competitive… the pathway Effectors may be feed-forward activators or Substrate inhibition or activation feedback inhibitors Kinetics are sigmoid ("S-shaped") The availability of substrates and cofactors usually determines how fast the reaction goes As product accumulates, the apparent rate of the enzymatic reaction will decrease General Features of Allosteric Regulation Allosteric activation/inhibition In most metabolic pathways there is at least 1 key enzyme Usually these pacemaker enzymes are in the first committed step in the pathway. Many times regulated by both feed forward and feedback mechanisms A B C D E Sigmoid v versus [S] plot. The dotted line represents the hyperbolic plot characteristic of normal Michaelis=Menten kinetics. 1 12/3/13 Allosteric activation/inhibition Reversible Covalent In most metabolic pathways there is at
    [Show full text]
  • Glucokinase Regulatory Protein As the Allosteric Switch for Glucokinase
    Molecular basis for the role of glucokinase regulatory protein as the allosteric switch for glucokinase Jung Min Choia,1, Moon-Hyeong Seoa,1, Hyun-Ho Kyeonga, Eunkyung Kima,2, and Hak-Sung Kima,b,3 aDepartment of Biological Sciences and bGraduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea Edited* by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved May 6, 2013 (received for review January 9, 2013) Glucokinase (GK) is a monomeric allosteric enzyme and plays between GK and GKRP and a modulation through the effectors a pivotal role in blood glucose homeostasis. GK is regulated by GK (17, 18). Despite the intensive studies on GK and GKRP, the regulatory protein (GKRP), and indirectly by allosteric effectors of molecular basis for the allosteric regulation mechanism of GK by GKRP. Despite the critical roles of GK and GKRP, the molecular basis GKRP remains poorly understood because of the lack of struc- for the allosteric regulation mechanism of GK by GKRP remains tural information of the GK/GKRP complex. unclear. We determined the crystal structure of Xenopus GK and Here, to demonstrate the molecular mechanism for the allo- GKRP complex in the presence of fructose-6-phosphate at 2.9 Å. steric regulation of GK by GKRP and effectors, we determined GKRP binds to a super-open conformation of GK mainly through the crystal structure of a Xenopus laevis GK and GKRP complex hydrophobic interaction, inhibiting the GK activity by locking a small in the presence of F6P. Structural analysis of the complex and domain of GK.
    [Show full text]
  • Post-Translational Modifications of the Energy Guardian AMP-Activated
    International Journal of Molecular Sciences Review Post-Translational Modifications of the Energy Guardian AMP-Activated Protein Kinase Ashley J. Ovens 1,2 , John W. Scott 2,3,4 , Christopher G. Langendorf 3, Bruce E. Kemp 2,3, Jonathan S. Oakhill 1,2 and William J. Smiles 1,* 1 Metabolic Signalling Laboratory, St Vincent’s Institute of Medical Research, School of Medicine, University of Melbourne, Fitzroy, VIC 3065, Australia; [email protected] (A.J.O.); [email protected] (J.S.O.) 2 Mary MacKillop Institute for Health Research, Australian Catholic University, Fitzroy, VIC 3000, Australia; [email protected] (J.W.S.); [email protected] (B.E.K.) 3 Protein Chemistry & Metabolism, St Vincent’s Institute of Medical Research, School of Medicine, University of Melbourne, Fitzroy, VIC 3065, Australia; [email protected] 4 The Florey Institute of Neuroscience and Mental Health, Parkville, VIC 3052, Australia * Correspondence: [email protected] Abstract: Physical exercise elicits physiological metabolic perturbations such as energetic and ox- idative stress; however, a diverse range of cellular processes are stimulated in response to combat these challenges and maintain cellular energy homeostasis. AMP-activated protein kinase (AMPK) is a highly conserved enzyme that acts as a metabolic fuel sensor and is central to this adaptive response to exercise. The complexity of AMPK’s role in modulating a range of cellular signalling cascades is well documented, yet aside from its well-characterised regulation by activation loop phosphorylation, AMPK is further subject to a multitude of additional regulatory stimuli. There- fore, in this review we comprehensively outline current knowledge around the post-translational Citation: Ovens, A.J; Scott, J.W; modifications of AMPK, including novel phosphorylation sites, as well as underappreciated roles for Langendorf, C.G; Kemp, B.E; Oakhill, ubiquitination, sumoylation, acetylation, methylation and oxidation.
    [Show full text]
  • Allosteric Regulation of Proteins a Historical Perspective on the Development of Concepts and Techniques
    GENERAL ARTICLE Allosteric Regulation of Proteins A Historical Perspective on the Development of Concepts and Techniques Kabir H Biswas Allostery is a mechanism by which the activity of a large num- ber of proteins is regulated. It is manifested as a change in the activity, either ligand binding or catalysis of one site of a protein due to a ligand binding to another distinct site of the protein. The allosteric effect is transduced by a change in the structural properties of the protein. It has been tradition- ally understood using either the concerted MWC (Monod, Wyman and Changeux) model, or the sequential KNF (Kosh- Kabir H Biswas is currently a land, Nemethy and Filmer) model of structural changes. How- Senior Research Fellow at the Mechanobiology Institute, ever, allostery is fundamentally a thermodynamic process and National University of requires an alteration in the enthalpy or entropy associated Singapore. His interest lies in with the process. studying how proteins are regulated by chemical or physical factors and is Introduction currently studying the organization of protein The regulation of essential biological processes such as metabo- complexes on the cell membrane. lism, growth and reproduction is a hallmark of living systems. Cells – the fundamental units of life, employ a variety of mech- anisms to achieve an exquisite control over these processes, and regulation of the activity of proteins is one of the means to fine- tune cellular processes. One of the ways of controlling the activ- ity of proteins is to alter the absolute levels of the protein in a cell. This is achieved either by altering the levels of the mRNA coding for the protein, or by altering the stability of the protein in the cellular environment.
    [Show full text]
  • Identification of New Allosteric Sites and Modulators of Ache Through Computational and Experimental Tools
    Journal of Enzyme Inhibition and Medicinal Chemistry ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20 Identification of new allosteric sites and modulators of AChE through computational and experimental tools Carlos Roca, Carlos Requena, Víctor Sebastián-Pérez, Sony Malhotra, Chris Radoux, Concepción Pérez, Ana Martinez, Juan Antonio Páez, Tom L. Blundell & Nuria E. Campillo To cite this article: Carlos Roca, Carlos Requena, Víctor Sebastián-Pérez, Sony Malhotra, Chris Radoux, Concepción Pérez, Ana Martinez, Juan Antonio Páez, Tom L. Blundell & Nuria E. Campillo (2018) Identification of new allosteric sites and modulators of AChE through computational and experimental tools, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 1034-1047, DOI: 10.1080/14756366.2018.1476502 To link to this article: https://doi.org/10.1080/14756366.2018.1476502 © 2018 The Author(s). Published by Informa View supplementary material UK Limited, trading as Taylor & Francis Group. Published online: 06 Jun 2018. Submit your article to this journal Article views: 100 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ienz20 JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 2018, VOL. 33, NO. 1, 1034–1047 https://doi.org/10.1080/14756366.2018.1476502 RESEARCH PAPER Identification of new allosteric sites and modulators of AChE through computational and experimental tools aà aà a b b,c d Carlos
    [Show full text]
  • Whence Cometh the Allosterome?
    PERSPECTIVE Whence cometh the allosterome? Janet E. Lindsley† and Jared Rutter† Department of Biochemistry, University of Utah, 15 North Medical Drive East, Room 4100, Salt Lake City, UT 84112-5650 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 5, 2006 (received for review May 31, 2006) Regulation of cellular functions can be accomplished by many mechanisms, including transcriptional regulation, alternative splicing, translational regulation, phosphorylation and other posttranslational covalent modifications, degradation, localization, protein– protein interactions, and small-molecule allosteric effectors. Largely because of advances in the techniques of molecular biology in the past few decades, our knowledge of regulation by most of these mechanisms has expanded enormously. Regulation by small- molecule, allosteric interactions is an exception. Many of the best-known allosteric regulators were discovered decades ago, when we knew little about all of these other forms of regulation. Allostery is the most direct, rapid, and efficient regulatory mechanism to sense changes in the concentration of small molecules and alter cellular responses to maintain homeostasis. In this perspective, we present the argument that allosteric regulation is underappreciated in the systems biology world and that many allosteric effectors remain to be discovered. wo of the earliest examples of cules. It can be very rapid, on the order past 4 decades, whereas those describing allosteric regulation are the of the rate of diffusion (Ϸ108 MϪ1⅐sϪ1), allosteric regulation have not (see Fig. control of oxygen binding to and readily reversible. And unlike 1). Although scientists have described hemoglobin by protons (1) and changes in covalent protein modification transcriptomes, proteomes, interactomes, TAMP activation of glycogen phosphory- such as phosphorylation͞dephosphoryla- and kinomes, the ‘‘allosterome’’ is still a lase (2).
    [Show full text]
  • Allosteric Regulation of Mammalian Fructose-1,6-Bisphosphatase Yang Gao Iowa State University
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2013 Allosteric regulation of mammalian fructose-1,6-bisphosphatase Yang Gao Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Gao, Yang, "Allosteric regulation of mammalian fructose-1,6-bisphosphatase" (2013). Graduate Theses and Dissertations. 13124. https://lib.dr.iastate.edu/etd/13124 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 Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Allosteric regulation of mammalian fructose-1,6-bisphosphatase by Yang Gao A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Richard Honzatko, Major Professor Amy Andreotti Mark Hargrove Scott Nelson Guang Song Iowa State University Ames, Iowa 2013 Copyright © Yang Gao, 2013. All rights reserved. ii TABLE OF CONTENTS Abbreviations v Abstract vi Chapter I. General Introduction 1 Figure 8 Thesis Organization 10 References 11 Chapter II. Mechanism of Displacement of a Catalytically Essential Loop from the Active Site of Mammalian Fructose-1,6-bisphosphatase 16 Abstract 16 Introduction 17 Experimental procedures 18 Results 22 Discussion 27 Table 31 Figure 33 References 42 Chapter III. Water Molecules in the Central Cavity of Mammalian Fructose-1,6- bisphosphatase as a Determinant of the Quaternary State 48 Abstract 48 Introduction 49 Experimental procedures 51 Results 54 Discussion 58 Table 61 iii Figure 65 References 71 Chapter IV.
    [Show full text]
  • Chem331 Regulation and Metab Intro 2014
    Enzyme Regulation - What Factors Influence Enzymatic Activity? Principle means of regulating enzyme activity • Reversible, non-covalent (allosteric and simple-MM) – typically small molecules • Reversible, covalent • Protein-Protein interactions • Zymogen activation • Protein expression and degradation • Availability (both of enzyme and substrate) Reversible Noncovalent Simple activation and inhibition by small molecules – substrate, natural regulators of enzymes MM kinetics Km, Vmax – competitive, non competitive… Substrate inhibition or activation The availability of substrates and cofactors usually determines how fast the reaction goes As product accumulates, the apparent rate of the enzymatic reaction will decrease Reversible Noncovalent: Allosteric Action at "another site" Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors These effectors are usually produced elsewhere in the pathway Effectors may be feed-forward activators or feedback inhibitors Kinetics are sigmoid ("S-shaped") Reversible Covalent Examples include: phosphorylation - dephosphorylaton, lipid modification. This method is important because it does not alter the total amount of protein and it is easily reversed depending on cellular needs Covalent modification - Protein kinases Phosphorylation/dephosphorylation Most common method of reversible modification - activation and localization Up one-third of all cellular proteins are phosphorylated (so since there are ~30,000 genes, that would be ~10,000 phosphoproteins/organism, and perhaps
    [Show full text]
  • Regulatory Enzymes Lecture-1
    B.Sc. (H) Biochemistry Ist Year, IInd Sem Enzymes Regulatory Enzymes Lecture-1 Dr. Prabha Arya Assistant Professor Deshbandhu College Control of Enzyme acivity 1. Control of Enzyme availability 2. Control of Enzyme activity Control of enzyme availability: The amount of a given enzyme in a cell depends on both its rate of synthesis and its rate of degradation. Each of these rates is directly controlled by the cell. For example, E. coli grown in the absence of the disaccharide lactose lack the enzymes to metabolize this sugar. Within minutes of their exposure to lactose, however, these bacteria commence synthesizing the enzymes required to utilize this nutrient. Similarly, the various tissues of a higher organism contain different sets of enzymes, although most of its cells contain identical genetic information Control of enzyme activity An enzyme’s catalytic activity may be directly controlled through conformational or structural alterations. The rate of an enzymatically catalyzed reaction is directly proportional to the concentration of its enzyme–substrate complex, which, in turn, varies with the enzyme and substrate concentrations and with the enzyme’s substrate-binding affinity . The catalytic activity of an enzyme can therefore be controlled through the variation of its substrate-binding affinity Types of Regulation to change the enzyme activity In cellular metabolism, groups of enzymes work together in sequential pathways to carry out a given metabolic process, such as the multireaction breakdown of glucose to lactate or the multireaction synthesis of an amino acid from simpler precursors. In such enzyme systems, the reaction product of one enzyme becomes the substrate of the next.
    [Show full text]
  • Arabidopsis and Chlamydomonas Phosphoribulokinase Crystal Structures Complete the Redox Structural Proteome of the Calvin-Benson
    bioRxiv preprint doi: https://doi.org/10.1101/422709; this version posted February 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Classification: Biological Sciences: Plant Biology 2 3 Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox 4 structural proteome of the Calvin-Benson cycle 5 6 Short title: Insight into chloroplast PRK structure 7 8 Libero Gurrieria, Alessandra Del Giudiceb, Nicola Demitric, Giuseppe Falinid, Nicolae Viorel Pavelb, 9 Mirko Zaffagninia, Maurizio Polentaruttic, Pierre Crozete, Christophe H. Marchande, Julien Henrie, 10 Paolo Trosta, Stéphane D. Lemairee, Francesca Sparlaa,1, Simona Fermanid,1 11 12 aDepartment of Pharmacy and Biotechnology – FaBiT, University of Bologna, 40126 Bologna, Italy 13 bDepartment of Chemistry, University of Rome ‘Sapienza’, 00185 Rome, Italy 14 cElettra - Sincrotone Trieste, 34149, Basovizza - Trieste, Italy 15 dDepartment of Chemistry ‘G. Ciamician’, University of Bologna, 40126 Bologna, Italy 16 eInstitut de Biologie Physico-Chimique, UMR8226, CNRS, Sorbonne Université, 75005 Paris, 17 France 18 19 20 1To whom correspondence should be addressed: 21 Simona Fermani: 22 Address: Department of Chemistry ‘G. Ciamician,’ University of Bologna, Via Selmi 2 - 23 40126 Bologna, Italy 24 Phone: +39 051 2099475 25 Email address: [email protected] 26 Francesca Sparla: 1 bioRxiv preprint doi: https://doi.org/10.1101/422709; this version posted February 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Protein–Protein Interactions in the Allosteric Regulation of Protein Kinases Patricia Pellicena and John Kuriyan
    COSTBI-407; NO OF PAGES 8 Protein–protein interactions in the allosteric regulation of protein kinases Patricia Pellicena and John Kuriyan Protein–protein interactions involving the catalytic domain of protein interactions can bring about conformational protein kinases are likely to be generally important in the changes in the kinase domain, in this case resulting in regulation of signal transduction pathways, but are rather activation [4]. Since then, much effort has been focused sparsely represented in crystal structures. Recently on filling in structural information for the kinome [5], determined structures of the kinase domains of the mitogen- understanding how intramolecular interactions control activated protein kinase Fus3, the RNA-dependent kinase the kinase domain (e.g. in the Src [6,7] and Abl [8,9] PKR, the epidermal growth factor receptor and Ca2+/ tyrosine kinases), how the conformation of the activation calmodulin-dependent protein kinase II have revealed loop controls kinase activity [10,11], how peptide sub- unexpected and distinct mechanisms by which interactions strates are recognized [12,13] and how the modulation of with the catalytic domain can modulate kinase activity. kinase structure is reflected in the specificity of kinase inhibitors [14]. Addresses Department of Molecular and Cell Biology, Department of Chemistry and Howard Hughes Medical Institute, University of California, and Surprisingly little has been revealed so far at the detailed Division of Physical Biosciences, Lawrence Berkeley National structural level about how protein–protein interactions Laboratory, Berkeley, CA 94720, USA involving the kinase domain result in changes in kinase activity, with one prominent exception being the early Corresponding author: Kuriyan, John ([email protected]) analysis of the Cdk–cyclin interaction [4].
    [Show full text]