title: Rational design of enantioselective enzymes requires considerations of entropy.
abstract: Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta(R-S)DeltaH(++)) and entropy (Delta(R-S)DeltaS(++)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta(R-S)DeltaS(++) was in all cases equally significant as Delta(R-S)DeltaH(++) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++). Although the changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta(R-S)DeltaH(++) than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta(R-S)DeltaS(++).
authors.affiliation: Department of Biotechnology, Royal Institute of Technology, SE-100 44 Stockholm, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Ottosson
authors.forename: J C
authors.initials: JC
authors.lastname: Rotticci-Mulder
authors.forename: D
authors.initials: D
authors.lastname: Rotticci
authors.forename: K
authors.initials: K
authors.lastname: Hult
mesh_terms: D002175:Candida / Q000201:enzymology* / Q000235:genetics; D019277:Entropy; D004789:Enzyme Activation; D005656:Fungal Proteins; D045782:Hemiterpenes; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000439:Pentanols / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D015202:Protein Engineering; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D045782:Hemiterpenes; D000439:Pentanols; C009034:prenol; D008049:Lipase; C431349:lipase B, Candida antarctica