The use of enzymes for the site‐specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP‐catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving‐group peptide. Our metal‐complexation‐based strategy enables efficient peptide/protein labeling at the N‐ or C‐terminus with near‐equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein–protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable.

Chemoenzymatic protein and peptide modification is a powerful means of generating defined, homogeneous conjugates for a range of applications. However, the use of transpeptidases is limited by the need to prepare synthetic peptide conjugates to be ligated, bulky recognition tags remaining in the product, and inefficient substrate turnover. Here, we report a peptide/protein labeling strategy that utilizes a promiscuous, engineered transpeptidase to irreversibly incorporate diverse, commercially available amines at a C-terminal asparagine. To demonstrate the utility of this approach, we prepare a protein–drug conjugate, generate a genetically inaccessible C-to-C protein fusion, and site specifically label both termini of a single protein in sequential steps.