ACTIVITY AND MOBILITY OF SUBTILISIN IN LOW WATER-ORGANIC MEDIA - HYDRATION IS MORE IMPORTANT THAN SOLVENT DIELECTRIC

The relationship between hydration, catalytic activity and protein dyn amics was investigated for subtilisin Carlsberg in organic solvents wi th low water content. The organic media were cyclohexane, dichlorometh ane or acetonitrile, with controlled thermodynamic water activity (a(w )). Catalytic rate profiles showed the same dependence on a(w) for the three different solvents. The structural mobility of the enzyme in ai r and organic media was probed by proton solid-state NMR relaxation me asurements. Both spin-lattice relaxation time (T-1) and line width at half height (apparent spin-spin relaxation time (T-2)) were determined for protein which was exchanged and hydrated with D2O. We found NMR r elaxation was much more dependent on a(w) than medium identity (despit e very different dielectrics) showing that enzyme hydration is the pri mary determinant of mobility. Results suggest that initial hydration u p to a(w) 0.22 causes rigidification of part of the protein structure. As a(w) is increased further, enzyme mobility is found to increase. A bove a(w) 0.44, a large increase in the proportion of more mobile prot ons coincides with a steep rise in catalytic activity for the enzyme i n each of the solvents studied. (C) 1998 Elsevier Science B.V. All rig hts reserved.