Hydrolases, particularly proteases, belong to the EC class 3 enzymes. They utilize water molecules to catalyze bond cleavage, typically breaking down large molecules into smaller ones, such as proteases, esterases, and glycosidases.

Protein ligases, classified under EC class 6 enzymes, hold significant applications in protein semi-synthesis, peptide cyclization, and proteomics. However, existing chemical ligation methods can be inefficient or require harsh conditions. Engineered ligases offer a more efficient and specific tool by catalyzing the joining of two molecules through the formation of new chemical bonds.

This study aimed to convert the HRV-3C protease, a highly specific cysteine hydrolase, into a novel peptide ligase through rational design. Our primary strategy involved computationally engineering a hydrophobic active-site pocket to suppress the enzyme’s intrinsic hydrolytic activity by sterically hindering the access of nucleophilic water molecules.

This work underscores a critical challenge in enzyme engineering and suggests that future design strategies must pivot from passively excluding water to actively enhancing the binding affinity and nucleophilicity of the desired acceptor substrate, enabling it to kinetically outcompete the hydrolysis reaction.

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