Multimolecular proofreading overcomes the activity-fidelity trade-off

Accurate signal processing is essential for proper cell functions, and can be achieved through kinetic proofreading, where an enzyme undergoes sequential state transitions and irreversible deactivation to enable high fidelity. However, synthetically constructing a biological proofreading system has been hindered by the difficulty in engineering single molecular state transitions. Here, we designed a protein circuit that combines diffusion and endocytosis to enable kinetic proofreading at the multimolecular level, without the conservation of total enzymes implicitly assumed in classic kinetic proofreading. Simulations revealed a previously overlooked yet experimentally crucial trade-off between circuit activity and fidelity, and theoretical analysis confirmed it to be fundamental in all kinetic proofreading systems. By integrating self-activation and mutual inhibition mechanisms, the circuit overcomes this activity-fidelity trade-off within biologically plausible parameter regimes. Our results extend proofreading schemes from single enzymes to a multimolecular context, and represent a practical and generalizable strategy for constructing high-fidelity synthetic biological circuits. HIGHLIGHTSO_LIWe design a multimolecular and multicellular proofreading circuit C_LIO_LIA previously overlooked yet practically relevant trade-off arises between circuit activity and fidelity C_LIO_LIThe activity-fidelity trade-off is fundamental in all kinetic proofreading circuits C_LIO_LISelf-activation and mutual inhibition mechanisms collectively overcome the activity-fidelity trade-off C_LI