Structural and Catalytic Roles of the Disulfide Bonds Cys19–Cys154 and Cys134–Cys199 in Trypsin-like Proteases: Evolutionary Insights for Disulfide Bond Acquisition

Trypsin is one of the most extensively studied enzymes in biochemistry. However, little information is available on the role of the disulfide bonds to establish the correct conformation and enzyme activity during molecular evolution. To obtain this information, two additional disulfide bonds corresponding to those found in human trypsin were individually or simultaneously introduced into the trypsin-like protease cocoonase (<i>Bombyx mori</i>), which contains three consensus disulfide bonds, and structural effects were analyzed. Enzyme assays of the mutant proteins revealed that, during molecular evolution, the Cys19-Cys154 bond contributed to improving substrate recognition (<i>K</i>_m), whereas the Cys134-Cys199 bond contributed to enhancing catalytic turnover (<i>k</i>_cat). In addition, the Cys134-Cys199 disulfide bond significantly increased the structural stability, whereas the Cys19-Cys154 disulfide bond promoted a more compact folded ensemble. Interestingly, when both disulfide bridges were introduced together, their effects acted synergistically, yielding the highest catalytic activity toward the substrate BAEE (<i>k</i>_cat/<i>K</i>_m). Taken together, these findings suggest that trypsin-like proteases evolved through a two-step adaptive process: an initial phase in which the catalytic efficiency (<i>k</i>_cat) and structural stability were enhanced, followed by a second phase in which the fold became more compact, thereby improving the overall enzymatic activity.