Catalysis of sequential reactions is often envisaged to occur by channeling of substrate between enzyme active sites without release into bulk solvent. However, while there are compelling physiological rationales for direct substrate transfer, proper experimental support for the hypothesis is often lacking, particularly for metabolic pathways involving RNA. Here, we apply transient kinetics approaches developed to study channeling in bienzyme complexes to an archaeal protein synthesis pathway featuring the misaminoacylated tRNA intermediate Glu-tRNA(Gln). Experimental and computational elucidation of a kinetic and thermodynamic framework for two-step cognate Gln-tRNA(Gln) synthesis demonstrates that the misacylating aminoacyl-tRNA synthetase (GluRS(ND)) and the tRNA-dependent amidotransferase (GatDE) function sequentially without channeling. Instead, rapid processing of the misacylated tRNA intermediate by GatDE and preferential elongation factor binding to the cognate Gln-tRNA(Gln) together permit accurate protein synthesis without formation of a binary protein-protein complex between GluRS(ND) and GatDE. These findings establish an alternate paradigm for protein quality control via two-step pathways for cognate aminoacyl-tRNA formation.
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