Enhancing recombinant urate oxidase stability and catalytic function with TMAO osmolyte: integrated experimental and computational approaches.

Compatible osmolytes like trimethylamine N-oxide (TMAO), known for preferentially stabilizing proteins in their folded states, offer a potential solution; however, the precise molecular mechanisms underlying their stabilizing effects are not fully understood. On the other hand, the poor thermal stability of pharmaceutical enzymes such as urate oxidase (UOX) in aqueous solutions remains a significant challenge. This study explored how TMAO modulates the enzyme's environment to enhance UOX's catalytic efficiency and stability. By combining experimental and computational approaches, we evaluated the kinetic, thermodynamic, and structural changes in urate oxidase with and without the osmolyte. Kinetic analysis revealed enhanced enzymatic activity in the presence of TMAO. Thermodynamic studies indicated negative values for enthalpy change (ΔH°) and entropy change (ΔS°), suggesting the involvement of van der Waals forces and hydrogen bonding in UOX-TMAO interactions. The negative value of free energy change (ΔG°) further confirms spontaneous binding. Fluorescence spectroscopy indicated structural modifications in the enzyme due to osmolyte binding, supported by a complex quenching mechanism. Molecular dynamics (MD) simulations demonstrated that TMAO increased UOX's structural compactness and enzyme stability, along with a rise in secondary structure content, which helped preserve the integrity of the active site. Moreover, molecular docking revealed a favorable binding of TMAO to UOX, primarily mediated by non-covalent interactions, thereby corroborating our experimental findings. These findings provide atomic insights into how TMAO can improve the stability and catalytic function of UOX.