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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 (ΔR‐SΔH‡) and entropy (ΔR‐SΔS‡) 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. ΔR‐SΔS‡ was in all cases equally significant as ΔR‐SΔH‡ 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 ΔR‐SΔH‡ and ΔR‐SΔS‡. Although the changes in ΔR‐SΔH‡ and ΔR‐SΔS‡ were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger ΔR‐SΔH‡ than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in ΔR‐SΔS‡.
Protein Science – Wiley
Published: Sep 1, 2001
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