The physicochemical bases for enzyme cold-adaptation remain elusive. The current view is that psychrophilic enzymes are often characterized by enhanced flexibility at low temperature to obtain higher catalytic efficiency, but the determinant behind this phenomenon is less well known. To shed light on the physicochemical bases for enzyme cold-adaptation, we conducted comparative molecular dynamics simulations on mesophilic proteinase K and its homologous psychrophilic counterpart. Results revealed that psychrophilic proteinase K had increased flexibility in regions near or opposite to active site or substrate-binding pocket. Comparison between the large concerted motions derived from essential dynamics (ED) analyses indicated that the degree of motion and direction of some regions in psychrophilic proteinase K could enlarge the substrate-binding pocket, thereby favoring catalytic efficiency and cold-adaptation. Free-energy calculations based on metadynamics simulations revealed a more "rugged" and complex free-energy landscape (FEL) for psychrophilic proteinase K than that for mesophilic proteinase K, implying that the former had richer conformational diversity. Comparison between the structural properties of the mesophilic and psychrophilic forms of proteinase K during MD simulations showed that the increased flexibility of the psychrophilic form resulted most probably from the reduced number of inter-atomic interactions and increased number of dynamic hydrogen bonds. A refined model of FEL was proposed to explain the effect of water molecules in facilitation of enzyme cold-adaptation.