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 atomistic mechanism underlying its concomitant effect on catalytic activity remains unclear. Here, we integrate molecular dynamics simulations, molecular docking, and biochemical assays to systematically investigate the structural-functional impacts of lysine acetylation. MD simulations revealed that acetylation induces global stabilization coupled with structural expansion and enhanced local flexibility. These perturbations propagate allosterically to the active site, resulting in its precise structural distortion. Biochemical assays demonstrated that acetylation trades a modest reduction in initial activity (~84% yield) for markedly enhanced autolytic resistance, with 80% of its activity retained after 6 hours. 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.