Molecular Understanding of Activity Changes of Alcohol Dehydrogenase in Deep Eutectic Solvents

Deep eutectic solvents (DESs) have emerged as promising solvents for biocatalysis. While their impact on enzyme solvation and stabilization has been studied for several enzyme classes, their role in substrate binding is yet to be investigated. Herein, molecular dynamics (MD) simulations of horse-liver alcohol dehydrogenase (HLADH) are performed in choline chloride-ethylene glycol (ChCl-EG) and choline chloride-glycerol (ChCl-Gly) at varying water concentrations. In the DES solutions, the active site was significantly constricted, and its flexibility reduced when compared to the aqueous medium. Importantly, the cavity size follows a similar trend as the catalytic activity of HLADH and as such explains previously observed activity changes. To understand the impact on the binding of the substrate (cyclohexanone), an umbrella sampling (US) setup was established to calculate the free energy changes along the substrate binding tunnel of HLADH. The US combined with replica exchange and NADH in its cofactor pocket provided the best sampling of the entire active site, explaining why the cyclohexanone binding on HLADH is reduced with increasing DES content. As different components in these multicomponent mixtures influence the substrate binding, we additionally applied the US setup to study the ability of the DES components to be present inside the substrate tunnel. The presented approach may become useful to understand enzyme behaviors in DESs and to enable the design of more enzyme-compatible and tunable solvents.