Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (a_w) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein‐bound water molecules increased linearly with a_w, until at a_w=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate‐binding site. At higher water activity, the thickness of the hydration shell increased up to 10 Å close to a_w=1. Above a limit of 1600 protein‐bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate‐binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive.