Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents

The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, a _w. The apparent turnover number A _cat ^app in these systems ranged from 0.2 to 80 s ^-1 and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing a _w. ¹⁹F NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on a _w. The rates of NMR-derived fast (k > 10 ⁷ s ^-1) and slow (k < 10 ⁴ s ^-1) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with k _cat. The ¹⁹F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant ε _as, which increased with hydration to ε _as ≈13 in each solvent. In both solvents, the transition state free energy data and ε _as followed Kirkwood's model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site's electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents.