Figure 4.

Cartoon depiction of the proposed mechanism of ATE1-catalyzed arginylation. (a) The canonical ATE1-catalyzed N-terminal arginylation reaction involves the transfer of Arg from the aminoacylated Arg-tRNA^Arg to the N terminus of the acceptor substrate to form a peptide bond in a nonribosomal manner. The inferred mechanism for this process involves nucleophilic attack of the 3′-acyl group by the N-terminal amino group, with subsequent release of the unmodified tRNA^Arg and the arginylated substrate. (b) An alternative ATE1-catalyzed reaction involves the transfer of Arg from the aminoacylated Arg-tRNA^Arg to the internal Asp/Glu side chain of the acceptor substrate to form an isopeptide bond in a nonribosomal manner. The postulated mechanism for this process involves the attack of the Arg-tRNA^Arg 3′-acyl group by the Asp/Glu carboxylate side chain, proton transfer at an active-site catalytic acid, release of the 3′-OH of the tRNA, and the formation of an unstable anhydride-like intermediate. The active-site conjugate base could then deprotonate the Arg α amine, attacking the anhydride carbonyl internally, releasing the Arg α carboxylate, and forming the isopeptide bond. In both panels, ATE1 is represented as the lavender shape, tRNA^Arg is represented as the gray shape, and the acceptor substrate protein is represented as the blue shape.