A460etukansi.fm Page 1 Friday, May 26, 2006 11:40 AM A 460 OULU 2006 A 460 UNIVERSITY OF OULU P.O. Box 7500 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA A SERIES EDITORS SCIENTIAE RERUM Prasenjit Bhaumik NATURALIUM Prasenjit Bhaumik Prasenjit ASCIENTIAE RERUM NATURALIUM Professor Mikko Siponen PROTEIN CRYSTALLOGRAPHIC BHUMANIORA STUDIES TO UNDERSTAND Professor Harri Mantila THE REACTION MECHANISM CTECHNICA Professor Juha Kostamovaara OF ENZYMES: DMEDICA α Professor Olli Vuolteenaho -METHYLACYL-CoA RACEMASE AND ESCIENTIAE RERUM SOCIALIUM Senior assistant Timo Latomaa ARGININOSUCCINATE LYASE FSCRIPTA ACADEMICA Communications Officer Elna Stjerna GOECONOMICA Senior Lecturer Seppo Eriksson EDITOR IN CHIEF Professor Olli Vuolteenaho EDITORIAL SECRETARY Publication Editor Kirsti Nurkkala FACULTY OF SCIENCE, DEPARTMENT OF BIOCHEMISTRY, BIOCENTER OULU, ISBN 951-42-8090-3 (Paperback) UNIVERSITY OF OULU ISBN 951-42-8091-1 (PDF) ISSN 0355-3191 (Print) ISSN 1796-220X (Online) ACTA UNIVERSITATIS OULUENSIS A Scientiae Rerum Naturalium 460 PRASENJIT BHAUMIK PROTEIN CRYSTALLOGRAPHIC STUDIES TO UNDERSTAND THE REACTION MECHANISM OF ENZYMES: α-METHYLACYL-COA RACEMASE AND ARGININOSUCCINATE LYASE Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Kuusamonsali (Auditorium YB210), Linnanmaa, on June 6th, 2006, at 12 noon OULUN YLIOPISTO, OULU 2006 Copyright © 2006 Acta Univ. Oul. A 460, 2006 Supervised by Professor Rik Wierenga Reviewed by Doctor Anette Henriksen Professor Reijo Lahti ISBN 951-42-8090-3 (Paperback) ISBN 951-42-8091-1 (PDF) http://herkules.oulu.fi/isbn9514280911/ ISSN 0355-3191 (Printed ) ISSN 1796-220X (Online) http://herkules.oulu.fi/issn03553191/ Cover design Raimo Ahonen OULU UNIVERSITY PRESS OULU 2006 Bhaumik, Prasenjit, Protein crystallographic studies to understand the reaction mechanism of enzymes: α-methylacyl-CoA racemase and argininosuccinate lyase Faculty of Science, Department of Biochemistry, University of Oulu, P.O.Box 3000, FI-90014 University of Oulu, Finland, Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014 University of Oulu, Finland Acta Univ. Oul. A 460, 2006 Oulu, Finland Abstract Enzymes catalyze chemical changes in biological systems. Therefore, to understand the chemistry of living systems, it is important to understand the enzyme structure and the chemistry of the enzyme's functional groups which are involved in catalysis. In this study, structure and function relationships of two enzymes, (1) α-methylacyl-CoA racemase from Mycobacterium tuberculosis (MCR) and (2) argininosuccinate lyase from Escherichia coli (eASL) have been studied using X-ray crystallography. The main focus of this study has been understanding the structure-function relationship of MCR. The eASL has been crystallized from a highly concentrated sample of purified recombinant α-methylacyl- CoA racemase in which it occurred as a minor impurity. The structure of eASL has been solved using molecular replacement at 2.44 Å resolution. The enzyme is a tetramer, but in this crystal form there is a dimer in the asymmetric unit. Each active site is constructed from loops of three different subunits. One of these catalytic loops, near residue Ser277 and Ser278, has been disordered in the previous structures of active lyases, but is very well ordered in this structure in one of the subunits due to the presence of two phosphate ions in the respective active site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex and therefore this structure has provided new information on the reaction mechanism of this class of enzymes. α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of α-methyl-branched CoA esters. An Amacr homologue from the eubacteria Mycobacterium tuberculosis, referred to as MCR, was taken as a model protein. MCR was purified, crystallized and the structure of unliganded protein was determined at 1.8 Å resolution using the MIRAS procedure. The structure shows that the enzyme is an interlocked dimer. To understand the reaction mechanism and the mode of substrate binding, several crystallographic binding studies were done using both wild type MCR and mutant H126A MCR crystals. In particular, the structures of the wild type MCR-complexes with (R, S)-ibuprofenoyl-CoA (1.85 Å), (R)-2- methylmyristoyl-CoA (1.6 Å) and (S)-2-methylmyristoyl-CoA (1.7 Å) were important in this respect. These crystal structures show that Asp156 and His126 are the two catalytic residues which are involved in proton donation and abstraction, respectively; when the (S)-enantiomeric substrate is bound in the active site and vice versa when the (R)-enantiomeric substrate is bound. The tight geometry of the active site also shows that His126 and Asp156 are involved in stabilizing the transition state. These crystal structures show that in the active site of MCR, there is one binding pocket for the CoA part and there are two different binding pockets (R-pocket and S-pocket) connected by a hydrophobic methionine rich surface for binding the fatty acyl part of the substrate. After substrate binding, proton abstraction takes place which produces a planar intermediate. Then, donation of a proton to the other side of the planar intermediate changes the configuration at the chiral center. During the stereochemical interconversion of the two enantiomers, the acyl group moves between R-pocket and S-pocket by sliding over the hydrophobic surface connecting these two pockets. Keywords: α-methylacyl-CoA racemase, argininosuccinate lyase, CoA transferase, proton transfer, racemase To Shrii Shrii Anandamurti Acknowledgements This work was carried out in the Department of Biochemistry and Biocenter Oulu at University of Oulu, during years 2001-2006. Financial support was from University of Oulu, Biocenter Oulu graduate school fellowship, and Tauno Tönningin Research Foundation. Firstly, I would like to warmly thank Professor Rik Wierenga for his excellent supervision, constant enthusiasm and support during this study. When I started my PhD research project I did not have any background in protein crystallography. However, he has been very patient to teach me both theoretical and practical aspects of protein crystallography. During the years of my thesis, there have been several ups and downs in my research, but he has always provided me enthusiasm and encouragement to make new discoveries. His patience, unquenchable curiosity and love for the research have been a great inspiration to me, and his continual support and encouragement have enablead me to complete my PhD thesis. I am also grateful to Professor Kalervo Hiltunen for his collaboration in this project, inspiration and helpful attitude during these years. I also wish to express my sincere thanks to Dr. Petri Kursula, who has been a great tutor and helped me a lot at the beginning of this research project. He has been a great inspiration to me during these years. I would like to thank also the other professors and group leaders of our department for creating such excellent research facilities and enjoyable working atmosphere. I would like to express my sincere thanks to Dr. Werner Schmitz and Professor Ernst Conzelmann from Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany for their collaboration in this project and giving me an opportunity to work in their laboratory. Dr. Schmitz has been very helpful and provided several interesting compounds for crystallographic binding studies. I would like to thank Dr. Päivi Pirilä for her effort to synthesize important ligands for this research project. I wish to thank all my co-authors for their contributions, especially Dr. Kalle Savolainen for his mutagenesis studies and Dr. Ulrich Bergmann for his help in mass spectrometric analysis of proteins. I am grateful to Dr. Anette Henriksen and Professor Reijo Lahti for their valuable comments on the manuscript of the thesis. I also thank the members of my thesis committee, especially Docent Tuomo Glumoff for his constructive criticism and comments which have helped me to carry out the research in the right direction. I would like to thank all the former and present members of RW group for everyday collaboration and many cheerful moments. I would like to specially thank Ville Ratas for his help at the beginning of this project. I wish to thank Dr. Antti Haapalainen for sharing tension and lots of fun during several synchrotron trips. I would like to thank Dr. Inari Kursula, Dr. Anu Mursula, Dr. Kristian Koski, Jukka Taskinen, Mikko Salin, Viivi Majava, Gitte Meriläinen, Markus Alahuhta, Mira Pekkala and Sanna Partanen for their help, company during these years. I wish to extend my sincere thanks to all the staff of our department. I want to thank Virpi Hannus, Anneli Kaattari, Pia Askonen and Tuula Koret for their kind help with many practical tasks. I would like to thank Kyösti Keränen and Jaakko Keskitalo for maintaining the X-ray equipment in proper condition for the datacollection during these years (especially in the summer time!). Jyrki Hänninen, André Juffer, Miki Kallio and Ari-Pekka Kvist are also acknowledged for setting up and maintaining computer systems. I sincerely thank all the people of the “Indian and Asian community” who have given me company and have always helped me during my stay in Oulu. I would like to thank Dr. Mahesh Somani, Satyan Sharma and Sharat Khungar for their friendship. I would like to specially thank my very good friend Dr. Baylie Damtie for his friendship and help. I also wish to express my big and special thanks to my very close and old friends Ranada, Saurabhda and Saurabhda’s wife Gargee for their support and help. It would have been very difficult to complete my dissertation without the help and support of my family members and close relatives. I would like to express my sincere thanks to my father and mother for their love, support and encouragement. I would like to thank my brother Surajit (Sintu) for his support and cheers.