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Peptide bond formation in living cells occurs at the peptidyl transferase center (PTC) of the large ribosomal subunit and involves the transfer of the peptidyl group from peptidyl-tRNA to aminoacyl-tRNA. Despite numerous kinetic and theoretical studies, many details of this reaction -such as whether it proceeds via a stepwise or concerted mechanism- remain unclear. In this study, we calculated the geometry and energy of the transition states and intermediates in peptide bond formation in the PTC environment using the ONIOM (our own n-layered integrated molecular orbital and molecular mechanics) method. The calculations indicated that the energy of the transition states of stepwise mechanisms are lower than those of concerted mechanisms and suggested that the reaction involves a neutral tetrahedral intermediate that is stabilized through the hydrogen-bonding network in the PTC environment. The results will lead to a better understanding of the mechanism of peptidyl transfer reaction, and resolve fundamental questions of the steps and molecular intermediates involved in peptide bond formation in the ribosome.

The search for the mechanism of ribosomal peptide bond formation is still ongoing. Even though the actual mechanism of peptide bod formation is still unknown, the dominance of proton transfer in this reaction is known for certain. Therefore, it is vital to take the quantum mechanical effects on proton transfer reaction into consideration; the effects of which were neglected in all previous studies. In this study, we have taken such effects into consideration using a semi-classical approach to the overall reaction mechanism. The M06-2X density functional with the 6-31++G(d,p) basis set was used to calculate the energies of the critical points on the potential energy surface of the reaction mechanism, which are then used in transition state theory to calculate the classical reaction rate. The tunnelling contribution is then added to the classical part by calculating the transmission permeability and tunnelling constant of the reaction barrier, using the numerical integration over the Boltzmann distribution for the symmetrical Eckart potential. The results of this study, which accounts for quantum effects, indicates that the A2451 ribosomal residue induces proton tunnelling in a stepwise peptide bond formation.

In this work, we have investigated a novel distal proton shuttle mechanism of ribosome catalyzed peptide bond formation reaction. The reaction was found to follow a two-step mechanism. A distal water molecule located about 6.1 Å away from the attacking amine plays as a proton acceptor and results in a charge-separated intermediate that is stabilized by the N terminus of L27 and the A-site A76 5'-phosphate. The ribose A2451 bridges the proton shuttle pathway, thus plays critical role in the reaction. The calculated 27.64 kcal•mol-1 free energy barrier of the distal proton shuttle mechanism is lower than that of eight-membered ring transition state. The distal proton shuttle mechanism studied in this work can provide new insights into the important biological peptide synthesis process.

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids., (© 2016 The Authors.)

The condensation (polymerization by water elimination) of molecular building blocks to yield the first active biopolymers (e.g. of amino acids to form peptides) during primitive Earth is an intriguing question that nowadays still remains open since these processes are thermodynamically disfavoured in highly dilute water solutions. In the present contribution, formation and hydrolysis of glycine oligopeptides occurring on a cluster model of sanidine feldspar (001) surface have been simulated by quantum mechanical methods. Results indicate that the catalytic interplay between Lewis and Brønsted sites both present at the sanidine surface, in cooperation with the London forces acting between the biomolecules and the inorganic surface, plays a crucial role to: i) favour the condensation of glycine to yield oligopeptides as reaction products; ii) inhibit the hydrolysis of the newly formed oligopeptides. Both facts suggest that mineral surfaces may have helped in catalyzing, stabilizing and protecting from hydration the oligopeptides formed in the prebiotic era.

Catalysis is an essential feature of living systems biochemistry, and probably, it played a key role in primordial times, helping to produce more complex molecules from simple ones. However, enzymes, the biocatalysts par excellence, were not available in such an ancient context and so, instead, small molecule catalysis (organocatalysis) may have occurred. The best candidates for the role of primitive organocatalysts are amino acids and short random peptides, which are believed to have been available in an early period on Earth. In this review, we discuss the occurrence of primordial organocatalysts in the form of peptides, in particular commenting on reports about seryl-histidine dipeptide, which have recently been investigated. Starting from this specific case, we also mention a peptide fragment condensation scenario, as well as other potential roles of peptides in primordial times. The review actually aims to stimulate further investigation on an unexplored field of research, namely one that specifically looks at the catalytic activity of small random peptides with respect to reactions relevant to prebiotic chemistry and early chemical evolution. [ABSTRACT FROM AUTHOR]

Abstract: From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized. Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with respect to temperature and pH. Among hydrolytic activities toward d-aminoacyl-pNAs, the enzymes showed strict specificity toward d-Phe-pNA, but showed broad specificity toward d-aminoacyl esters. The specific activity for d-Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As a second function, each enzyme showed peptide bond formation activity by its function of aminolysis reaction. Based on results of d-Phe–d-Phe synthesis under various conditions, we propose a reaction mechanism for d-Phe–d-Phe production. Furthermore, the enzymes exhibited peptide elongation activity, producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which revealed that the primary structure of each enzyme showed 30–60% identity with those of peptidases belonging to the clan SE, S12 peptidase family categorized as serine peptidase with d-stereospecificity. [Copyright &y& Elsevier]

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNAimet compared to the one for the elongator tRNAs indicates that only the initiator tRNAimet specifies the amino acid side chain. [ABSTRACT FROM AUTHOR]

The ribosome catalyzes two fundamental biological reactions: peptidyl transfer, the formation of a peptide bond during protein synthesis, and peptidyl hydrolysis, the release of the complete protein from the peptidyl tRNA upon completion of translation. The ribosome is able to utilize and distinguish the two different nucleophiles for each reaction, the αα-amine of the incoming aminoacyl tRNA versus the water molecule. The correct binding of substrates induces structural rearrangements of ribosomal active-site residues and the substrates themselves, resulting in an orientation suitable for catalysis. In addition, active-site residues appear to provide further assistance by ordering active-site water molecules and providing an electrostatic environment via a hydrogen network that stabilizes the reaction intermediates and possibly shuttles protons. Major questions remain concerning the timing, components, and mechanism of the proton transfer steps. This review summarizes the recent progress in structural, biochemical, and computational advances and presents the current mechanistic models for these two reactions. [ABSTRACT FROM AUTHOR]

Abstract: The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well. [Copyright &y& Elsevier]

Ribosome-catalyzed peptide bond formation is a crucial function of all organisms. The ribosome is a ribonucleoprotein particle, with both RNA and protein components necessary for the various steps leading to protein biosynthesis. Evolutionary theory predicts an early environment devoid of complex biomolecules, and prebiotic peptide synthesis would have started in a simple way. A fundamental question regarding peptide synthesis is how the current ribosome-catalyzed reaction evolved from a primitive system. Here we look at both prebiotic and modern mechanisms of peptide bond formation and discuss recent experiments that aim to connect these activities. In particular, RNA can facilitate peptide bond formation by providing a template for activated amino acids to react and can catalyze a variety of functions that would have been necessary in a pre-protein world. Therefore, RNA may have facilitated the emergence of the current protein world from an RNA or even prebiotic world. [ABSTRACT FROM AUTHOR]

In the ribosome, the decoding and peptide bond formation sites are composed entirely of ribosomal RNA, thus confirming that the ribosome is a ribozyme. Precise alignment of the aminoacylated and peptidyl tRNA 3'-ends, which is the major enzymatic contribution of the ribosome, is dominated by remote interactions of the tRNA double helical acceptor stem with the distant rims of the peptidyl transferase center. An elaborate architecture and a sizable symmetry-related region within the otherwise asymmetric ribosome guide the A→P passage of the tRNA 3'-end by a spiral rotatory motion, and ensures its outcome: stereochemistry suitable for peptide bond formation and geometry facilitating the entrance of newly formed proteins into their exit tunnel. [ABSTRACT FROM AUTHOR]

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3′ end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation. [ABSTRACT FROM AUTHOR]