Uncovering N‐Glycosylation‐Mediated Allosteric Activation Coupled With Stability Design Enabling High‐Yield Human Enterokinase Production

ABSTRACT Glycosylation, a prevalent post‐translational modification, regulates numerous cellular processes and modulates protein activity. Proteinases are the most widely researched enzymes, many of which were glycosylated in eukaryotes. Serine protease enteropeptidase used in pharmaceuticals and biotechnology industry, due to its specifical recognition and clean cleaves of the D 4 K polypeptide sequence, are N‐glycosylated at four sites. In this study, we focused on glycosylation and deglycosylation engineering of human light‐chain enteropeptidase (HLEK) to obtain more efficient mutants. By creating a series of site‐directed deglycosylation mutants, we found that glycosylation sites Asn103 and Asn165 that are remote from the catalytic center, significantly enhanced HLEK's enzymatic activity. Molecular dynamics simulations combined with Protein Residue Dynamical Network (PRDN) analysis revealed that glycosylation shortens signal‐transmission paths across the residue network, providing a mechanistic basis for the observed k cat enhancement. Specifically, presence of N‐glycosylation shortens the residues signal‐transmission paths, thereby increasing k cat . As the fully deglycosylated HLEK (N64Q/N103Q/N125Q/N165Q), suffered from low expression, we performed ΔΔ G guided design, the generated mutants demonstrated 76.5‐fold increase in expression levels but 9.7‐fold decrease in catalytic activities. Re‐introduction of N‐gycosylation at 103 and 165 to the designed mutant restored allosteric activation and yielded a substantially improved total enzyme output. In fed‐batch fermentation, this HLEK mutant reached a total enzyme activity of 5.86 × 10 6 ± 2.52 × 10 5 U L −1 (358 mg/L, four times the highest level previously reported). Our results show that distal N‐glycosylation can allosterically accelerate catalysis via remodeling of residue interaction networks, and that combining N‐glycosylation‐guided mechanistic insights with stability engineering offers a robust route to high‐yield, high‐activity protease production.