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Enzymatic Studies of Alcohol Dehydrogenase by a Combination of in Vitro and in Silico Methods

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Enzymatic Studies of Alcohol Dehydrogenase by a Combination of in Vitro and in Silico Methods From the Department of Medical Biochemistry and Biophysics Karolinska Institutet, Stockholm, Sweden ENZYMATIC STUDIES OF ALCOHOL DEHYDROGENASE BY A COMBINATION OF IN VITRO AND IN SILICO METHODS Mikko Hellgren Stockholm 2009 1 All previously published papers are reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Reproprint AB © Mikko Hellgren, 2009 ISBN 978-91-7409-567-8 Printed by 2 2009 Gårdsvägen 4, 169 70 Solna Abstract The family of alcohol dehydrogenases (ADHs) catalyzes conversions of alcohols, ketons and aldehydes. The early discovery and isolation of ADH (1937) was followed by numerous investigations. It was also the first dimeric enzyme for which the three- dimensional structure was determined (1974). Recent findings have revealed new physiological functions for the ADH enzymes. One type is a key enzyme in hepatic retinol metabolism, another is a main formaldehyde scavenger and a regulator of S- nitrosothiols levels. ADH genes have been shown to be connected to diseases and syndromes, such as alcoholism and asthma. Hence, investigations of structure-function relationships of the ADH enzymes are of both physiological and medical interest. The aim of this thesis was to study structure-function relationships and to investigate and identify ligands for ADH, with in vitro and in silico methods. The catalytic activities of human, mouse and rat ADH2 for retinoids, were determined. The Km values for human ADH2 are the lowest among all known human dehydrogenases, which supports a key role for human ADH2 in the hepatic retinoid metabolism. ADH3 is an enzyme with a proposed role as an NO scavenger. Two new lines of ligands, bile acids and fatty acids, were investigated for their potential effects on NO homeostasis. The bronco dilatatory effect of NO suggests that ADH3 inhibition could potentially work as treatment of obstructive lung disorders. The stability of the quaternary structure of sorbitol dehydrogenase (SDH) was determined by in vitro experiments and in silico energy calculations. A hydrogen-bonding network crucial for the tetrameric stability in SDH was identified. This network is located at a region enclosing the structural zinc site in mammalian ADHs. The structural zinc site was studied in detail by a combination of molecular dynamics and quantum mechanics simulations. The simulations revealed that the interaction between the cysteine residues and the zinc atom is of an electrostatic and covalent nature. With in silico and in vitro simulations, interactions between ligands and the active site were determined, revealing site specific interactions within both ADH2 and ADH3. Furthermore, studies of subunit interactions and the structural zinc site revealed properties of the quaternary stability. 3 List of original papers This thesis is based on the following papers, printed in the appendix, and will be referred to in the text by their roman numerals. Publications are sorted by publication dates in chronological order: I. Hellgren M, Strömberg P, Gallego O, Martras S, Farrés J, Persson B, Parés X, Höög JO (2007) Alcohol dehydrogenase 2 is a major hepatic enzyme for human retinol metabolism. Cellular and Molecular Life Sciences 64(4): 498- 505 II. Hellgren M, Kaiser C, de Haij S, Norberg A, Hoog JO (2007a) A hydrogen- bonding network in mammalian sorbitol dehydrogenase stabilizes the tetrameric state and is essential for the catalytic power. Cellular and Molecular Life Sciences 64(23): 3129-3138 III. Brandt EG, Hellgren M, Brinck T, Bergman T, Edholm O (2009) Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site. Physical Chemistry Chemical Physics 11(6): 975-983 IV. Staab CA, Hellgren M, Grafström RC, Höög JO (2009) Medium-chain fatty acids and glutathione derivatives as inhibitors of S-nitrosoglutathione reduction mediated by alcohol dehydrogenase 3. Chemico-Biological Interactions 180(1): 113-118 V. Hellgren M., Carlsson J., Östberg L., Staab C.A., Persson B., Höög J.O. Virtual screening for ligands to human alcohol dehydrogenase 3. Manuscript 4 Additional publications not included in the thesis 1. Hellgren M, Sandberg L, Edholm O (2006) A comparison between two prokaryotic potassium channels (KirBac1.1 and KcsA) in a molecular dynamics (MD) simulation study. Biophysical Chemistry 120(1): 1-9 2. Staab CA, Hellgren M, Höög JO (2008) Dual functions of alcohol dehydrogenase 3: implications with focus on formaldehyde dehydrogenase and S-nitrosoglutathione reductase activities. Cellular and Molecular Life Sciences 65(24): 3950-3960 5 Contents 1 Introduction to alcohol dehydrogenase (ADH)........................................................11 1.1 A brief historical background: From the Big Bang to the discovery of ADH ..11 1.2 Medium-chain dehydrogenase/reductase (MDR) proteins...............................13 1.3 The MDR-ADH protein superfamily...............................................................14 1.4 Human ADH1 to ADH5..................................................................................15 1.4.1 ADH1......................................................................................................16 1.4.2 ADH2......................................................................................................16 1.4.3 ADH3......................................................................................................17 1.4.4 ADH4......................................................................................................18 1.4.5 ADH5......................................................................................................18 1.5 Tetrameric ADHs ............................................................................................18 1.6 Sorbitol dehydrogenase ...................................................................................19 1.7 The central dogma of ADH .............................................................................20 1.7.1 Genes and protein sequences ...................................................................20 1.7.2 Protein expression patterns......................................................................21 1.7.3 Tertiary structure .....................................................................................22 1.7.4 Active site................................................................................................23 1.7.5 Structural zinc site ...................................................................................25 1.7.6 Quaternary structure ................................................................................25 1.8 The metabolism of endogenous and exogenous substrates ..............................27 1.8.1 Catalytic activities ...................................................................................28 1.8.2 Ethanol ....................................................................................................29 1.8.3 Formaldehyde..........................................................................................30 1.8.4 Nitric oxide..............................................................................................31 1.8.5 Vitamin A................................................................................................32 1.8.6 Sorbitol and the polyol pathway ..............................................................33 1.8.7 Coenzyme NAD ......................................................................................34 1.9 Mutations, polymorphisms and disease ...........................................................35 2 Theories and methods for enzymatic studies ...........................................................37 2.1 Proteins as enzymes.........................................................................................37 2.2 In vitro methods for protein experiments.........................................................38 2.2.1 Expression, purification and identification of proteins ............................38 2.2.2 Gelfiltration .............................................................................................38 2.2.3 Enzyme kinetics.......................................................................................39 2.3 In silico methods for protein interaction..........................................................40 2.3.1 Molecular dynamics simulations with GROMACS.................................41 2.3.2 Monte Carlo simulations with ICM .........................................................42 2.3.3 Quantum chemistry simulations with GAUSSIAN..................................43 3 Results and discussions, Paper I to V ......................................................................44 3.1 Paper I .............................................................................................................44 3.2 Paper II............................................................................................................46 3.3 Paper III...........................................................................................................48 3.4 Paper IV ..........................................................................................................50 6 3.5 Paper V............................................................................................................52 4 Conclusions.............................................................................................................54 5 Reflections ..............................................................................................................55
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