Reversible peptide tagging system to regulate enzyme activity
Yao Chen, Yi Shi, Dengming Ming, He Huang, Ling Jiang
Abstract
Peptide tagging systems are widely used across scientific fields. Here, to obtain a peptide tagging system with high covalent linkage efficiency, we rationally designed the ReverseTag/ReverseCatcher system, building upon the ester-bond-based peptide tagging system Cpe0147 439 −563 /Cpe0147 565−587 . Our optimized system exhibits a second-order rate constant of 1.92 ± 0.002 × 10 4 M −1 s −1 , a 21.7-fold increase over the wild-type system. The ester bond between ReverseTag and ReverseCatcher demonstrates exceptional mechanical stability, withstanding forces over 2 nN, while remaining hydrolyzable under alkaline conditions with significant energy input. To leverage this for enzymatic catalysis, we introduced a T11S mutation into ReverseCatcher, allowing pH-controlled reversible bond formation and cleavage. Applying this strategy to exo -inulinase (EXINU) enables precise, pH-dependent enzyme activity control. This sustainable method for modulating enzyme-catalyzed reactions offers broad biotechnological potential.