Pseudomonas cepacia lipase (PCL) is a highly enantioselective catalyst in the hydrolysis or formation of esters. It shows high enantioselectivity towards esters of chiral primary alcohols. Several scientists have tried to explain this enantioselectivity via modelling studies, however their results are in disagreement. / To explain PCL enantioselectivity towards chiral primary alcohols, we coupled structural studies and modelling of PCL-transition state analogue complexes containing pure enantiomers of chiral primary alcohols as alcohol moieties. / We first elucidate the different enantiospecificity of the enzyme for primary and secondary alcohols showing that primary alcohols do not bind to PCL like secondary alcohols. We then show that PCL enantioselectivity towards 3-methyl-2-phenyl-1-propanol, a chiral primary alcohol without oxygen at the stereocentre, rests on the better hydrogen bond of the fast enantiomer with catalytic histidine and that there are only subtle binding differences between the two alcohol enantiomers within the catalytic pocket of PCL. / Next, we show that PCL enantioselectivity towards chiral primary alcohols having oxygen at the stereocentre depends mainly on the interaction of the oxygen at the stereocentre with PCL residues Leu17 and Thr18, which may favour or disfavour one enantiomer depending on its structural features. This explains why a simple empirical rule cannot be reliably applied to this class of substrates. / We then exploit PCL interfacial activation and the use of a solvent that stabilises the active open form of the enzyme to increase its enantioselectivity towards esters of 2-methyl-3-phenyl-l-propanol and 2-phenoxy-l-propanol from 16 to ≥190 and from 17 to 70. / Last, we try to increase the enantioselectivity of Bacillus thermocatenulatus lipase (BTL2) towards solketal-n-octanoate and some other chiral primary alcohols by structure-guided saturation mutagenesis. However most of the mutants were inactive and we did not find a more enantioselective BTL2 mutant. / Overall, we have shown that lipase enantioselectivity towards chiral primary alcohols is always based on subtle enzyme-substrate interactions, hence the difficulty to increase lipases enantioselectivity towards these substrates by substrate and protein engineering.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84295 |
| Date | January 2003 |
| Creators | Mezzetti, Alessandra |
| Contributors | Kazlauskas, Romas J. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002091089, proquestno: AAINQ98327, Theses scanned by UMI/ProQuest. |
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