Dissecting the role of substrate folding in enzymatic digestion.

The efficiency of enzymatic proteolysis is often attributed to the properties of the enzyme itself, with the substrate typically viewed as a passive participant. In this study, we demonstrate that the conformational state of the substrate critically influences proteolytic efficiency. Using human serum albumin (HSA) as a model substrate, papain as the enzyme, and urea as a controlled denaturing agent, we systematically investigated how substrate conformation might affect proteolysis. While papain maintains its structural and functional integrity across varying urea concentrations, HSA transitions through well-defined conformational states (native, compact intermediate, and unfolded), allowing us an opportunity to isolate the effects of the substrate structure. Utilizing site-specific fluorescent labeling and single-molecule fluorescence correlation spectroscopy, we monitor the progression of proteolysis. Our results show that digestion slows at 3M urea, where HSA adopts a compact form, and accelerates at 6M, where HSA takes on an unfolded state, compared to native HSA. These results reveal that substrate folding critically influences the digestion kinetics, probably by controlling protease accessibility and underscoring its importance in mechanistic enzymology and proteomics workflows.