Activity and mobility of subtilisin in low water organic media: hydration is more important than solvent dielectric Johann Partridge *, Philip R. Dennison, Barry D. Moore, Peter J. Halling Department of Pure and Applied Chemistry, Royal College Building, University of Strathclyde, 204 George Street, Glasgow G1 1XW, UK Received 20 February 1998; accepted 21 April 1998 Abstract The relationship between hydration, catalytic activity and protein dynamics was investigated for subtilisin Carlsberg in organic solvents with low water content. The organic media were cyclohexane, dichloromethane 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 air and organic media was probed by proton solid-state NMR relaxation measurements. 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 D 2 O. We found NMR relaxation was much more dependent on a w than medium identity (despite very different dielectrics) showing that enzyme hydration is the primary determinant of mobility. Results suggest that initial hydration up 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. Above a w 0.44, a large increase in the proportion of more mobile protons coincides with a steep rise in catalytic activity for the enzyme in each of the solvents studied. ß 1998 Elsevier Science B.V. All rights reserved. Keywords : Activity ; Mobility ; Organic solvent ; Hydration 1. Introduction The study of enzyme activity in low water systems can provide interesting new insights into the under- lying mechanisms of biocatalysis. The activity of sol- id enzyme particles is observed to rise as the water content of the system is increased. Hydration also enhances the mobility of the protein structure, and it is widely believed that it is this which aids catalysis [1]. However, experimental evidence for this belief is rather limited, and sometimes contradictory. Accurate measurement of catalytic activity is di¤- cult in a purely solid-state system. Suspending the enzyme in a suitable organic solvent provides a me- dium for the transfer of substrates and products, allowing straightforward assay of activity [2^9]. Such systems are of great biotechnological interest. The catalytic activity of enzymes in organic media is known to depend strongly on the amount of water associated with the enzyme [10]. However, the rela- tionship between protein dynamics, hydration and catalytic activity remains to be de¢nitively estab- lished. A wide variety of methods have been used to probe protein structure and dynamics. Unfortu- nately, many of them cannot be applied to solid par- ticles of enzymes in low-water media, particularly 0167-4838 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII:S0167-4838(98)00086-7 * Corresponding author. Fax: +44 (141) 553-1161; E-mail : j.partridge@strath.ac.uk Biochimica et Biophysica Acta 1386 (1998) 79^89