Crystallographic Studies on the Structure-Function Relationships in Triosephosphate Isomerase
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CRYSTALLOGRAPHIC STUDIES INARI ON THE STRUCTURE-FUNCTION KURSULA RELATIONSHIPS IN Biocenter Oulu and Department of Chemistry, TRIOSEPHOSPHATE ISOMERASE University of Oulu OULU 2003 INARI KURSULA CRYSTALLOGRAPHIC STUDIES ON THE STRUCTURE-FUNCTION RELATIONSHIPS IN TRIOSEPHOSPHATE ISOMERASE Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L10), Linnanmaa, on May 16th, 2003, at 12 noon. OULUN YLIOPISTO, OULU 2003 Copyright © 2003 University of Oulu, 2003 Supervised by Professor Rik Wierenga Reviewed by Doctor Kristina Djinovic Carugo PhD Mikael Peräkylä ISBN 951-42-7009-6 (URL: http://herkules.oulu.fi/isbn9514270096/) ALSO AVAILABLE IN PRINTED FORMAT Acta Univ. Oul. A 400, 2003 ISBN 951-42-7008-8 ISSN 0355-3191 (URL: http://herkules.oulu.fi/issn03553191/) OULU UNIVERSITY PRESS OULU 2003 Kursula, Inari, Crystallographic studies on the structure-function relationships in triosephosphate isomerase Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu; Department of Chemistry, University of Oulu, P.O.Box 3000, FIN-90014 University of Oulu Oulu, Finland 2003 Abstract The triosephosphate isomerase (TIM) barrel superfamily is a broad family of proteins, most of which are enzymes. At the amino-acid-sequence level, many of the members of this family share little, if βα any, homology. Yet, they adopt the same three-dimensional ( )8 fold. The TIM barrel fold seems to be a good framework for many different kinds of enzymes, providing unique possibilities for both natural and human-designed evolution, as the catalytic center and the stabilizing features are separated to different ends of the barrel. Indeed, in the light of most recent studies, it seems likely that at least most of the different TIM barrel enzymes, catalyzing a huge variety of reactions, have evolved from a common ancestor. TIM can be considered a real text-book enzyme — its catalytic properties and stucture-function relationships have been studied for decades. Still, at present, we are quite far from understanding the structural features that make TIM and other enzymes such superior catalysts in both efficiency and precision. TIM is a dimeric enzyme that consists of two identical subunits of 250 residues. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in glycolysis. The basics of this reaction are well known, but there is ongoing discussion about the details of the proton transfer steps, and three alternative pathways have been suggested. In addition, it is a fascinating question how the enzyme succeeds in abstracting a highly stable proton from a carbon atom of the substrate. This study was undertaken to shed light on some of the questions concerning the structure- function relationships in TIM. The most important findings are the elucidation of the role of Asn11 as a catalytic residue and the meaning of the flexibility of both the catalytic Glu167 side chain as well as the substrate during catalysis, and the presence of a low-barrier hydrogen bond between Glu167 and a transition-state analogue, 2-phosphoglycolate. Furthermore, significant results were obtained on the importance of a conserved salt bridge, 20 Å away from the active site and the dimer interface, for the stability and folding of TIM as well as on the factors influencing the opening of the flexible loop 6 upon product release. Keywords: catalysis, enzyme, low-barrier hydrogen bond, protein folding, proton abstraction For Petri, Ville, Lotta, and Jussi Acknowledgements This work was carried out at the Department of Biochemistry and Biocenter Oulu, University of Oulu, during the years 1998-2003. I owe deep gratitude to professor Rik Wierenga for his expertise and most kind supervision. I am also grateful to professor Kalervo Hiltunen and all the other professors, group leaders, and staff at the department for creating excellent working conditions. I would like to thank Dr. Kristina Djinovic and Dr. Mikael Peräkylä for their critical review of this manuscript and helpful suggestions, and Eliisa Kursula for careful revision of the language. I thank all my co-authors for their contributions, especially Dr. Annemie Lambeir for instructive discussions on enzyme kinetics. I wish to thank all the nice and helpful people at the synchrotrons I have visited - EMBL/DESY, ESRF, and MAX-Lab. I am grateful to all former and present members of the RW group for helping in many practical and theoretical matters, and for being such good company. Especially Anu, Gitte, Mira, Päivi, and Sanna have ensured that the days at work have not been all too serios, but also lots of fun. During these years, I have not had enough time to spend with friends. Still, I could always count on them being there. Besides being great company, Kati, Kaisa, Mika, Taru, and Katri have often been available for baby sitting. Jarmo and Sanna, despite living far from us, have meant a lot for our family. I am most grateful for the friendship of Kati and Petri, and their children Jaakko and Anni. I owe my warmest thanks to my parents, who always encouraged me to ask questions and raised the curiosity in me. I am also grateful to my little sister Taru for numerous moments of joy. Yet more, I want to thank my parents-in-law for helping whenever needed. Most of all people, I would like to thank my dear Petri for his love and support. Besides my husband and best friend, he has been my best teacher and collaborator. Our three wonderful children, Ville, Lotta, and Jussi, are the joy of my life and have always helped me remember what life really is about. This work was financially supported by the Finnish Academy and Suomalainen Konkordia-liitto. Oulu, May 2003 Inari Kursula Abbreviations ADP adenosine 5’-monophosphate ATP adenosine 5’-triphosphate C carboxyl CD circular dichroism DGAP D-glyceraldehyde-3-phosphate DHAP dihydroxyacetone phosphate E65Q the mutant Leishmania mexicana mexicana TIM, where Glu65 is replaced by Gln E. coli Escherichia coli FMNH reduced form of flavin adenine mononucleotide G3P glycerol-3-phosphate HisA N-[(5’-phosphoribosyl)formimino]-5-aminoimidazole-4- carboxamide ribonucleotide isomerase HisF imidazole glycerol phosphate synthase IPP 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate kDa kilodalton LBHB low-barrier hydrogen bond L. mexicana Leishmania mexicana mexicana N amino NAD oxidized form of nicotinamide adenine dinucleotide NADH reduced form of nicotinamide adenine dinucleotide NADP nicotinamide adenine dinucleotide phosphate NMR nuclear magnetic resonance PDB Protein Data Bank 3PG 3-phosphoglycolate PGA 2-phosphoglycolate PGH phosphoglycolohydroxamate Pi inorganic phosphate P. woesei Pyrococcus woesei Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis T. brucei Trypanosoma brucei brucei TIM triosephosphate isomerase TLS translation, libration, screw rotation T. maritima Thermatoga maritima Tris tris(hydroxymethyl)aminomethane TrpA the a -subunit of tryptophan synthase TrpC indole-3-glycerol phosphate synthase TrpF phosphoribosylanthranilate isomerase V. marinus Vibrio marinus YGGS the Tyr210-Gly211-Gly212-Ser213 region in loop 7 of TIM List of original articles This thesis is based on the following articles, referred to in the text by their Roman numerals. I Lambeir AM, Backmann J, Ruiz-Sanz J, Filimonov V, Nielsen JE, Kursula I, Norledge BV & Wierenga RK (2000) The ionisation of Glu65 buried at the monomer-monomer interface in Leishmana mexicana triosephosphate isomerase is thermodynamically linked to the stability of the dimer. Eur J Biochem 267: 2516-24. II Kursula I, Partanen S, Lambeir AM, Antonov DM, Augustyns K & Wierenga RK (2001) Structural determinants for ligand binding and catalysis of triosephosphate isomerase. Eur J Biochem 268: 5189-5196. III Kursula I, Partanen S, Lambeir AM & Wierenga RK (2002) The importance of the conserved Arg191-Asp227 salt bridge of triosephosphate isomerase for folding, stability, and catalysis. FEBS Lett 518: 39-42. IV Kursula I & Wierenga RK (2003) Crystal structure of triosephosphate isomerase complexed with 2-phosphoglycolate at 0.83-Å resolution. J Biol Chem 278: 9544-9551. Contents Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction.................................................................................................................15 2 Review of the literature..............................................................................................16 2.1 The TIM barrel superfamily of proteins..........................................................16 2.1.1 The TIM barrel structure..........................................................................16 2.1.2 Classification of TIM barrel proteins..................................................... 20 2.1.3 Evolution of TIM barrel proteins............................................................22 2.1.3.1 Convergent evolution towards a stable fold?.............................23 2.1.3.2 Evidence for divergent evolution from a common ancestor.... 24 2.1.3.3 TIM barrels in different genomes................................................. 26 2.1.4 Folding and stability of TIM barrel proteins........................................ 26 2.1.5 Engineering the TIM barrel fold.............................................................28 2.2 Triosephosphate isomerase...............................................................................28