PMC

Single-turnover progress curves for α-phosphorothioate-substituted adenylate and 2′- and 3′-substituted variants at tRNA A76 using ThrRS wild-type enzyme. (A) R_p (filled squares) and S_p (filled circles) α-phosphorothioate-substituted ATP analogs. (B) Modified A76 tRNAs (2′-dA76-, 3′-dA76-, and tRNA^Thr-periodate-treated). The 3′-dA76 (open squares) and tRNA^Thr-periodate treated (filled triangles) served as the controls for the reaction.

(A) X-ray crystal structure of ribosomal substrates bound in the active site [PDB entry 1VQN (78)]. The α-NH₃ group of the A-site substrate has been replaced with a hydroxyl group to inhibit the reaction. The A76 2'-OH hydrogen bonds with the α-NH₃, and the 2'- and 3'-OHs coordinate a solvent molecule on their far side. (B) Schematic of the peptidyl transfer intermediate and the proposed mechanism for resolution into products. The 2'-OH acts as a proton shuttle by simultaneously donating a proton to the 3'-OH and accepting a proton from the α-NH₃. (C) X-ray crystal structure of the transition state mimic CCA-phosphate-puromycin_Phe-CC [PDB entry 1VQP (78)]. The non-bridging phosphate oxygens mimic the peptide and the oxyanion. The proposed proton transfers are indicated with arrows.

The mechanism of aminoacyl transfer mediated by the 2′-OH of tRNA A76 in E. coli ThrRS. Dotted lines represent H-bonds, and the arrows indicate the proton relay. See Discussion for the detailed explanation of the mechanism.

(A) Catalytic water (W1) is activated by the 2′-OH. (B) A proton transfer occurs from W1 to N3 of A76 via the 2′-OH and W2 to form a protonated A76 intermediate. (C) A proton is subsequently transferred to O3′ via W2 and 2′-OH to form the hydrolyzed product. The oxyanionic intermediate is stabilized by the GXXXP loop. The reaction coordinate refers to bond broken (Ala-C(O) – O3′-A76) – bond formed (Ala-C(O) – OH₂). Panels A, B and C show the optimized reactant, intermediate, and products, respectively. (D) The calculated potential energy surface for this mechanism at the BP86/TZVPP//BP86/SV(P) level of theory for the QM domain.