A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation

Abstract

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction. Herein, we report a method to make PAL-mediated intermolecular ligation irreversible by coupling it to glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation. In this method, the acyl donor substrate of PALs is designed to have glutamine at the P1′ position of the Asn-P1′-P2′ tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group in which the exposed N-terminal glutamine is cyclized by QC, quenching the Gln Nα-amine in a lactam. Using this method, PAL-mediated ligation can achieve near-quantitative yields even at an equal molar ratio between the two ligation partners. We have demonstrated this method for a wide range of applications, including protein-to-protein ligations. We anticipate that this cascade enzymatic reaction scheme will make PAL enzymes well suited for numerous new uses in biotechnology.