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

TL;DR

This paper reveals how acetylation makes trypsin more resistant to self-destruction while slightly reducing its activity.

Contribution

The study identifies the molecular mechanism by which acetylation stabilizes trypsin and alters its active site structure.

Findings

Acetylation increases trypsin's stability, retaining 80.78% activity after six hours of autolysis.

Acetylation causes structural changes that slightly misalign catalytic residues and alter substrate binding.

Allosteric effects from acetylation propagate to the active site, distorting its geometry.

Abstract

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.

•Lysine acetylation enhances trypsin's autolytic resistance at the expense of a modest reduction in activity.•Acetylation induces global structural stabilization coupled with local flexibility and active site distortion.•Allosteric propagation of perturbations disrupts the catalytic triad geometry.•A catalytically suboptimal substrate-binding mode with altered hydrogen bonding is formed in acetylated trypsin.

Lysine acetylation enhances trypsin's autolytic resistance at the expense of a modest reduction in activity.

Acetylation induces global structural stabilization coupled with local flexibility and active site distortion.

Allosteric propagation of perturbations disrupts the catalytic triad geometry.

A catalytically suboptimal substrate-binding mode with altered hydrogen bonding is formed in acetylated trypsin.

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