Dynamic catalytic domain plasticity governs substrate specificity in industrial serine proteases: Structural and functional implications

Serine proteases play a crucial role in biocatalysis, where subtle conformational differences can significantly impact substrate binding and catalytic efficiency. In this study, proteinase K, protease 2709, and protease PB92 were analyzed through enzymatic kinetics, structural modeling, molecular docking, and molecular dynamics (MD) simulation. Kinetic data showed that proteinase K had the highest catalytic efficiency and substrate affinity, while PB92 exhibited the weakest activity. Although all three Enzymes shared the conserved Ser-His-Asp triad, notable differences in secondary structures, catalytic site geometry, and substrate-binding pockets were observed. Proteinase K and 2709 featured stable catalytic domains and similar triad configurations, whereas PB92 had a more flexible, disordered active site. Docking and MD results indicated strong substrate binding and structural stability for proteinase K and 2709, with PB92 undergoing significant conformational shifts. Binding energy decomposition revealed that proteinase K and 2709 relied on hydrogen bond-driven recognition via structural catalytic core (SCC) residues, while PB92 primarily engaged in hydrophobic interactions from peripheral regions. These findings offer mechanistic insight into how structural differences among serine proteases govern their catalytic behavior and substrate recognition, providing a foundation for future enzyme engineering and drug design efforts.

Interaction; Serine proteases; Structural catalytic core.