Influence of osmolytes and denaturants on the structure and enzyme activity of α-chymotrypsin

Enzymes are very sensitive and highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of cosolvents, the fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways, and alternatively, enzymes can be stabilized or destabilized. To understand the contribution of osmolytes and denaturants on the stabilization, related to the associated structural changes and enzyme activity of α-chymotrypsin (CT), we have monitored differential scanning calorimeter (DSC), circular dichroism (CD), enzyme activity, and gel electrophoresis as a function of osmolyte or denaturant concentration. The present investigation compares the compatibility of osmolytes and deleterious effects of denaturants on the structure, function, and enzyme activity of CT. This comparison has provided new important insight on the contribution of cosolvent effects on protein folding/unfolding, enzyme activity, and understanding of protein-solvent interactions. Our DSC results reveal that the enthalpy change (ΔH) and Gibbs free energy of change (ΔG_u) of CT in osmolyte (trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, and sucrose) increase linearly as osmolyte concentration increases, while those values decrease sharply in the presence of denaturants (urea and guanidine hydrochloride (GdnHCl)). The modifications in the secondary structure of this β/β protein, as quantified by the CD spectra, showed reasonable enhancement for β-strands in the presence of the osmolytes as compared to buffer, which contributes to its stabilization power. Evidently, we observed that naturally occurring osmolytes have a dominant contribution to the stabilization of CT while not enhancing its enzyme activity. In contrast, our results revealed that the denaturants enhanced the surface of the enzyme by binding to the surface of CT, which leads to zero enzyme activity.