Recent progress in elucidating the peptide bond formation process on the ribosome has led to notion of a proton shuttle mechanism where the 2'-hydroxyl group of the P-site tRNA plays a key role in mediating proton transfer between the nucleophile and leaving group, whereas ribosomal groups do not actively participate in the reaction. Despite these advances, the detailed nature of the transition state for peptidyl transfer and the role of several trapped water molecules in the peptidyl transferase center remain major open questions. Here, we employ high-level quantum chemical ab initio calculations to locate and characterize global transition states for the reaction, described by a molecular model encompassing all the key elements of the reaction center. The calculated activation enthalpy as well as structures are in excellent agreement with experimental data and point to feasibility of an eight-membered "double proton shuttle" mechanism in which an auxiliary water molecule, observed both in computer simulations and crystal structures, actively participates. A second conserved water molecule is found to be of key importance for stabilizing developing negative charge on the substrate oxyanion and its presence is catalytically favorable both in terms of activation enthalpy and entropy. Transition states calculated both for six-and eight-membered mechanisms are invariably late and do not involve significant charge development on the attacking amino group. Predicted kinetic isotope effects consistent with this picture are similar to those observed for uncatalyzed ester aminolysis reactions in solution.density functional theory | protein synthesis T he peptide bond formation step in protein synthesis is catalyzed by the peptidyl transferase center (PTC) on the large ribosomal subunit. This reaction involves the attack of the α-amino group of an aminoacyl-tRNA molecule bound to ribosomal A-site on the ester carbon of the peptidyl-tRNA in the adjacent P-site of the ribosome. The growing peptide chain is thereby transferred to the A-site tRNA and elongated by one amino acid. Recent years have witnessed considerable progress in our understanding of the peptidyl transfer process due to high-resolution crystallographic structures of the large ribosomal subunit with transition state (TS) analogs (1), as well as kinetic measurements (2-9), mutagenesis data (4, 10-14), and computational studies (15)(16)(17). Hence, the current model of the peptidyl transfer reaction is that ribosomal nucleobases are not directly involved in bond making or breaking through acid-base catalysis (4,10,11,13,18,19), but that the A76 2′-OH group of the P-site substrate plays a key role in mediating proton transfer from the attacking nucleophile to the leaving 3′ ester oxygen (1,15,(20)(21)(22). Furthermore, it was shown that the lower free energy barrier for the ribosome reaction, compared to an uncatalyzed reference reaction in water, is entirely due to a less negative activation entropy (3, 6, 7). Besides contributions from substrate proximity and...