Molecular basis of trypsin's autolysis resistance acetylation for sustained enzymatic activity

Acetylation serves as an effective strategy to enhance trypsin's resistance to autolysis, yet the underlying molecular mechanism remains unclear. Integrating molecular dynamics (MD) simulations and biochemical assays, we show that acetylation induces global stabilization (RMSD decreased by 0.03 nm) coupled with structural expansion (Rg increased by 0.01 nm) and a significant (p < 0.05) increase in local flexibility. These perturbations propagate allosterically to the active site, resulting in its precise structural distortion. Experimentally, acetylated trypsin exhibited markedly improved stability, retaining 80.78% of its activity after six hours of autolysis versus only 54.2% for the native enzyme, despite an initial activity reduction of 23.2%. The molecular basis for this trade-off is an allosterically rewired state that enhances structural integrity while slightly misaligning catalytic residues and promoting a low-efficiency substrate binding mode. Collectively, our work provides atomic-level insights useful for rationally designing trypsin variants with optimized performance in food enzyme engineering.