Changing the residues interaction pattern as a universal mechanism for enzyme inactivation and denaturation in supercritical CO2

Abstract

Enzyme-catalyzed reactions in supercritical CO2 conditions bear many biotechnological advantages. However, many enzymes cannot function in this dense fluid due to the possible inactivation and denaturation. The molecular mechanism of enzyme inactivation in supercritical CO2 has been very controversial due to the limitations of experiments to examine macromolecule structure at the molecular levels in supercritical conditions. Most of hypotheses insist on the unique role of lysine residues in enzyme denaturation in supercritical CO2. Here, using molecular dynamics simulations and lysozyme as a model enzyme, we challenged this approach. Through the examination of structural properties of the different residues, it was explored for the first time that lysine is not the only responsible of the denaturation mechanism. Based on the simulation results, the denaturation process occurs by escaping of many surface charged and polar residues from the solvent and forming non-native H bonds with the other residues of the enzyme in a cooperative process. This changes the interaction pattern of the enzyme residues in supercritical CO2. In fact, the denaturation process is mainly due to the many new non-native H bonds of surface charged and polar residues and is not a result of single residue instability. The new interactions lead to the destruction of many essential native H bonds of the enzyme. We have evaluated this mechanism by simulations of lipase and peroxidase as two enzymes with the different activity and stability in supercritical CO2. Structural analysis, solvent accessible surface area, and H bond numbers confirm that both enzymes show similar behavior to lysozyme in supercritical CO2, although with the different extents. Lipase showed lower change of solvent accessible surface area and fewer new H bonds in comparison to peroxidase, which are in line with the experimental observations of more stability and activity of this enzyme in comparison to peroxidase in supercritical CO2. Moreover, both enzymes show normal behavior with minimal changes and new interactions in the aqueous conditions. We think that this new mechanism can explain the reasons of enzyme denaturation and justify the different behaviors of the enzymes in supercritical CO2 solvent. It was also revealed that creating more interactions among the polar and charged residues at the enzyme surface and increasing the surface hydrophobicity could be effective ways in stabilizing the enzymes in supercritical CO2.