Enzyme substrate solvent interactions A case study on serine hydrolases Linda Fransson Doctoral thesis Royal Institute of Technology School of Biotechnology Stockholm 2008 © Linda Fransson 2008 In tihs pdf file, I have corrected typos and layout errors present in the printed version. Royal Institute of Technology School of Biotechnology AlbaNova University Center SE-106 91 Stockholm Sweden ISBN 978-91-7415-094 TRITA-BIO-Report 2008:15 ISSN 1654-2312 Printed in Stockholm, August 2008 E-PRINTAB, Lästmakargatan 24 111 44 Stockholm ABSTRACT Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further, hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated. Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate speci- ficity was favoured by enthalpic stabilisation. Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solvent molecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour. In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified. SAMMANFATTNING Reaktionshastighet och specificitet har uppmätts för transacylering av fettsyra- estrar i lösningsmedel där lipas B från Candida antarctica och cutinas från Humicola insolens har använts som katalysatorer. För dessa enzymer kan den traditionella vattebaserade enzymologin utökas till att även omfatta studier i lösningsmedel, vilket ger möjlighet att erhålla stor variation i interaktionsenergier mellan enzym, substrat och omgivning. Vidare studerades även hydrolytiska reaktioner katalyserade av Bacillus subtilis. Termodynamisk analys av experimentaldata visade att enzymers bidrag till acceleration av reaktionshastighet jämfört med motsvarande syrakatalyserade reaktion hade ett entropiskt ursprung. Samtidigt visade studier av skillnader i aktiveringsentropi och -entalpi mellan enantiomerer och homologa estrar att hög substratspecificitet gynnades av entalpisk stabilisering. Lösningsmedel hade en tydlig påverkan på såväl enzymaktivitet som -specificitet. Skillnader i löslighet substrat emellan påverkar specificiteten då substratbindning är en jämvikt mellan enzymbundet substrat och substrat i fri lösning. Dessutom visade sig lösningsmedel kunna inhibera enzymer både kompetitivt och icke-kompetitivt. Flera homologa dataserier uppvisade en mycket god entalpi-entropi- kompensation. Ett eventuellt fysikaliskt innehåll dränks dock lätt av samvarier- ande fel. Av de kompensatoriska relationer som identifierats i den här avhandlingen visade det sig i ett fall vara möjligt att säkerställa en relation som inte var dominerad av statistiska fel. I övriga fall kunde ingen sådan slutsats dras. LIST OF ARTICLES This thesis is based on the following articles which are referred to by their roman numerals. I Ottosson J, Fransson L, Hult K: Substrate entropy in enzyme enantioselectivity: An experimental and molecular modeling study of a lipase. Protein Sci 2002, 11(6): 1462-1471. II Ottosson J, Fransson L, King JW, Hult K: Size as a parameter for solvent effects on Candida antarctica lipase B enantioselectivity. Biochim Biophys Acta, Protein Struct Molec Enzym 2002, 1594(2): 325-334. III Graber M, Irague R, Rosenfeld E, Lamare S, Fransson L, Hult K: Solvent as a competitive inhibitor for Candida antarctica lipase B. Biochim Biophys Acta, Proteins Proteomics 2007, 1774(8): 1052-1057. IV Leonard V, Fransson L, Lamare S, Hult K, Graber M: A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol. ChemBioChem 2007, 8(6): 662-667. V Kourist R, Bartsch S, Fransson L, Hult K, Bornscheuer UT: Understanding promiscuous amidase activity of an esterase from Bacillus subtilis. ChemBioChem 2008, 9(1): 67-69. VI Fransson L, Bernhardt P, Hult K: On the benefit of an active site. Manuscript. TABLE OF CONTENTS Enzyme substrate solvent interactions ........................................................................................................ 1 The origin of enzyme catalytic power ........................................................................................................... 2 Proximity effects ...................................................................................................................................................... 2 The role of steric hindrance in enzyme specificity ............................................................................ 9 Structural basis for specificity maxima ....................................................................................................... 11 On solvent effects on enzymatic catalysis .............................................................................................. 15 Substrate solubility as a basis for solvent effects.................................................................................... 15 Solvent as a competitive inhibitor................................................................................................................. 18 Solvent as a non-competitive inhibitor....................................................................................................... 19 Solvent stabilisation of transition state ....................................................................................................... 23 Correlation between solvent effects and physical parameters ......................................................... 25 Enthalpy-entropy compensation .................................................................................................................... 27 Appendix A – Derivation of rate equations for dead-end and mixed-type inhibition .............................................................................................................................................................................. 34 Dead-end (competitive) inhibition ............................................................................................................... 34 Mixed-type inhibition ......................................................................................................................................... 35 Acknowledgements .................................................................................................................................................... 37 Enzyme substrate solvent interactions ENZYME SUBSTRATE SOLVENT INTERACTIONS Enzymes show an intriguing ability of performing highly specific and efficient catalysis. Their catalytic performance is governed by the interactions between substrate, enzyme and its surrounding. With the development of enzyme- catalysed synthesis enzymes have been introduced into organic solvents. This has opened a new research area within enzymology which allows a large variation of reaction media. In this thesis, enzyme catalytic efficiency and specificity will be discussed in terms of molecular interactions between enzymes, substrates and the surrounding solvent. Transacylation reactions catalysed by Candida antarctica lipase B and Humicola insolens cutinase have been used as a model system in all but one case, where hydrolysis reactions catalysed by Bacillus subtilis esterase were studied. In total, the following reactions have been investi- gated: Paper I O Solvent R1 O R1 CALB Hexane OH O + O 2 + O 1 R 6 Solvent R2 6 R =CH3 or C2H5 2 R =C2H5, C3HCHCH3 or C(CH3)3 Paper II O Solvent O Supercritical CO2 OH CALB + O + Decaline Hexane O Solvent O Cyclopentane 1,4-Dioxane 6 6 Tetrahydrofuran Acetone Dichloromethane Carbon disulfide Paper III O CALB O Effector OH + + 2-Pentanone OH 3-Pentanone O O 2-Methyl-2-pentanol 3-Methyl-3-pentanol 2-methylpentane 3-methylpentane Paper IV O Effector OH Water O CALB O + + O OH Paper V O N O N Solvent 2 O BSE2 O 2 H2O + + Water Water X HO XH X=O or NH Paper VI O CALB, O Solvent cutinase or + Hexane O HCl O OH + n n OH Toluene n=2-10 Solvent n=2-10 Acetonitrile 1 The origin of enzyme catalytic power THE ORIGIN OF ENZYME CATALYTIC POWER Several hypotheses have been put forward on how enzymes achieve their increased reaction rate compared to uncatalysed reactions.1,2 One of the most well-established hypotheses on enzyme catalytic ability is the concept of transition-state stabilisation.3 It was introduced by Linus Pauling in 1948 sug- gesting that enzymes exert catalytic power by being complementary to the reactant transition state. Together with Eyring´s transition