Biophysical insights into osmolytes-driven enhancements in urate oxidase activity and stability

The thermal instability of pharmaceutical enzymes such as uricase or urate oxidase (UOX) in liquid solutions is a major problem. Notably, compatible osmolytes are a specific class of osmolytes that can preferentially stabilize the folded state of proteins. However, the detailed molecular interactions that enable them to affect protein stability are still not completely elucidated. This study aimed to investigate how the enzyme environment could be altered using osmolytes to improve its catalytic activity and stability. Initially, experimental conditions were optimized using response surface methodology (RSM). Then, kinetic and thermodynamic properties, as well as structural changes of urate oxidase, were assessed using spectroscopic and computational techniques in the presence and absence of mixed osmolytes. Kinetic parameters indicated an improvement in the catalytic function of urate oxidase and the results of thermodynamic analysis indicated that the van der Waals forces and hydrogen bonding network played a crucial role in the UOX-osmolytes interactions. Fluorescence measurements suggested that osmolyte-UOX interactions alter the enzyme's structure. The data revealed a complex quenching mechanism between the enzyme and osmolyte. Molecular dynamics (MD) simulations showed an increase in stability and structural compactness of the enzyme in the presence of mixed osmolytes. Furthermore, it depicted an increase in the abundance of secondary structure contents of the enzyme, which in turn maintained the integrity of its active site. Also, the molecular docking analysis further supported the experimental results. These findings revealed mechanisms by which binary-compatible osmolytes could have relevant effects on the catalytic function and stability of urate oxidase.