HYDRATION ENERGY LANDSCAPE OF THE ACTIVE-SITE CAVITY IN CYTOCHROME P450CAM

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules tha t, upon ligand binding, are either displaced into bulk solvent or reta ined to mediate protein-ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to id entify the water molecules involved in the binding process when ligand s bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active s ite, displacing partially disordered solvent, and the protein is well characterized experimentally, We calculated the free energy difference s for having five to eight water molecules in the active site cavity o f the unliganded enzyme from molecular dynamics simulations by thermod ynamic integration employing a three-stage perturbation scheme. The co mputed free energy differences between the hydration states are small (within 12 kJ mol-(1)) but distinct, Consistent with the crystallograp hic determination and studies employing hydrostatic pressure, we calcu lated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381-396, 1998. (C) 19 98 Wiley-Liss, Inc.