It has been suggested that evolutionary adaptation has adjusted enzyme conformational mobility as a key parameter allowing optimum catalysis at a given temperature. 1 For example, the concept of "corresponding states" 1c for homologous enzymes from mesophilic and thermophilic organisms states that enzyme topology, flexibility, and mechanism are comparable at the temperature where the enzymes display optimum activity. 1b We investigated whether this concept would hold true for enzymatic catalysis in organic solvents. We demonstrate that the highest enzyme activity was displayed when the structure and conformational mobility was most similar to that of the enzyme in aqueous buffer. This suggests that the evolutionary adaptation concept 1 may indeed be applicable to enzyme activity and activation in neat organic solvents.For dehydrated enzyme powders suspended in organic solvents molecular lubricants 2 are critical for achieving an optimum enzyme activity. 3 For example, adjustment of the level of residual enzyme bound water is important for attaining optimum enzyme activity in organic solvents, 4 which strongly suggests a relationship between enzyme activity and conformational mobility in organic solvents. 3,4e This is also supported by enzyme activation with denaturing cosolvents, such as DMSO and dimethyl formamide, 5 and crown ethers 6 and cyclodextrins. 7 We have demonstrated that the latter macrocyclic compounds increase the conformational mobility of the suspended enzyme. 6e,7a Identification of factors contributing to optimum enzyme activity in organic solvents is notoriously difficult. 3,6e,7a,b This is due to the fact that the drop in activity when compared to the aqueous environment may stem from changes in protein structure and dynamics, energetics of substrate desolvation and transition state stabilization, among other. 3 We pursued the strategy to vary the conformational mobility and structure of subtilisin Carlsberg in 1,4-dioxane by (a) co-drying with various concentrations of the crown ether 18-crown-6 and (b) addition of DMSO. Thus, we only changed one experimental parameter in a set of Supporting Information Available: (1) Calculation of the spectral correlation coefficient, second derivative spectra of subtilisin films (2) with 18-crown-6 and (3) in 1,4-dioxane/DMSO at various temperatures, (4) plots of the spectral correlation coefficient vs temperature used to determine the T d values, (5) tables with the correlation coefficients for the various subtilisin films prior to and after solvent exposure at various temperatures (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. The formation of the S product ester 9,10 in the reaction of sec-phenethyl alcohol with vinyl butyrate 6e,7a,b was studied for subtilisin co-dried with increasing concentrations of 18-crown-6. After initial enzyme activation ( Figure 1A) with an optimum activity at the 0.7 mass ratio (crown ether-to-subtilisin), the activity dropped. Such curves have been reported before, 6d but ...

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (±)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V 0 , ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38°C and 56°C, respectively). No relationship between this property and V 0 was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V 0 was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of sub-tilisin activation by crown ethers, as recently suggested.