How does active site water affect enzymatic stereorecognition?

The E value for ketone reduction catalyzed by a secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus is temperature dependent, due to a relatively large activation entropy difference, ΔΔS‡, which favors the formation (or reaction) of the (R)-enantiomer of 2-butanol and 2-pentanol. In contrast, the activation enthalpy, ΔΔH‡, favors the formation of the (S)-enantiomer of 2-butanol and 2-pentanol. C295A mutant SADH shows very large reductions in ΔΔS‡ compared to wild-type SADH, in addition to the expected reductions in ΔΔH‡. The decrease in ΔΔH‡ can be readily explained on the basis of steric interaction and van der Waals contacts of the enantiomeric substrates in the active site. The comparison of ΔΔS‡ of the wild-type and C295A mutant SADH reduces the possible entropy contributions to those associated with the Cys-295 sulfhydryl group. Examination of the crystal structure shows an ordered water molecule in the small pocket, located at a distance of 4.1 Å from the sulfur atom of Cys-295. Thus, the stereospecificity of SADH may be at least partly determined by the selective expulsion of this bound water from the small alkyl binding pocket upon binding of large substituents in the small alkyl pocket. The data obtained with wild-type, S39T and C295A SADH with NADP, SNADP and APADP show a reasonable enthalpy–entropy compensation relationship, suggesting a contribution of solvation. Our results suggest that selective release of bound solvent from the active site upon binding of enantiomeric substrates may contribute to the differential activation entropy, ΔΔS‡.