Water dynamics and salt-activation of enzymes in organic media: mechanistic implications revealed by NMR spectroscopy.

Deuterium spin relaxation was used to examine the motion of enzyme-bound water on subtilisin Carlsberg co-lyophilized with inorganic salts for activation in different organic solvents. Spectral editing was used to ensure that the relaxation times were associated with relatively mobile deuterons, which were contributed almost entirely by D(2)O rather than hydrogen-deuteron exchange on the protein. The results indicate that the timescale of motion for residual water molecules on the biocatalyst, (tau(c))(D(2)O), in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% CsF) as (k(cat)/K(M))(app) of the biocatalyst preparation increased from 0.092 s(-1) x M(-1) (salt-free) to 1,140 s(-1) x M(-1) (98% CsF). A similar effect was apparent in acetone; the timescale decreased from 24 ns (salt-free) to 2.87 ns (98% KF), with a corresponding increase in (k(cat)/K(M))(app) of 0.140 s(-1) x M(-1) (salt-free) to 12.8 s(-1) x M(-1) (98% KF). Although a global correlation between water mobility and enzyme activity was not evident, linear correlations between ln[(k(cat)/K(M))(app)] and (tau(c))(D(2)O) were obtained for salt-activated enzyme preparations in both hexane and acetone. Furthermore, a direct correlation was evident between (k(cat)/K(M))(app) and the total amount of mobile water per mass of enzyme. These results suggest that increases in enzyme-bound water mobility mediated by the presence of salt act as a molecular lubricant and enhance enzyme flexibility in a manner functionally similar to temperature. Greater flexibility may permit a larger degree of local transition-state mobility, reflected by a more positive entropy of activation, for the salt-activated enzyme compared with the salt-free enzyme. This increased mobility may contribute to the dramatic increases in biocatalyst activity.