Catalysis Of Peptide Bond Formation Research Articles

Non-Stressful Death of 23S rRNA Mutant G2061C Defective in Puromycin Reaction

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.

An easy and effective demonstration of enzyme stereospecificity and equilibrium thermodynamics

Enzyme stereospecificity and equilibrium thermodynamics can be demonstrated using the coupling of two amino acid derivatives by Thermoase C160. This protease will catalyze peptide bond formation between Z-L-AspOH and L-PheOMe to form the Aspartame precursor Z-L-Asp-L-PheOMe. Reaction completion manifests itself by precipitation of the product. As the product has almost zero solubility, the equilibrium favors condensation and thus a normally hydrolytic enzyme catalyzes the opposite reaction. Neither Z-D-AspOH with L-PheOMe nor Z-L-AspOH with D-PheOMe produces any visible product.

Glycine Peptide Bond Formation Catalyzed by Faujasite

The catalysis of peptide bond formation between two glycine molecules on H-FAU zeolite was computationally studied by the M08-HX density functional. Two reaction pathways, the concerted and the stepwise mechanism, starting from three differently adsorbed reactants, amino-bound, carboxyl-bound, and hydroxyl-bound, are studied. Adsorption energies, activation energies, and reaction energies, as well as the corresponding intrinsic rate constants were calculated. A comparison of the computed energetics of the various reaction paths for glycine indicates that the catalyzed reaction proceeds preferentially via the concerted reaction mechanism of the hydroxyl-bound configuration. This involves an eight-membered ring of the transition structure instead of the four-membered ring of the others. The step from the amino-bound configuration to glycylglycine is the rate-determining step of the concerted mechanism. It has an estimated activation energy of 51.2 kcal mol(-1). Although the catalytic reaction can also occur via the stepwise reaction mechanism, this path is not favored.

An Alternative Mechanism for the Catalysis of Peptide Bond Formation by L/F Transferase: Substrate Binding and Orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.

Transient Domain Interactions in Non‐Ribosomal Peptide Synthetases

Non-ribosomal peptide synthetases (NRPSs) are multi-module, multi-domain enzymes, synthesizing a wealth of important natural products in bacteria and fungi. These products often find pharmaceutical applications as antibiotics, antitumor agents or immunosuppressant drugs. NRPS use a remarkable assembly line organization to covalently load substrates into each module and catalyze peptide bond formation between substrates loaded on adjacent modules. The substrates may optionally be modified by tailoring domains. These multiple catalytic steps require a series of sequential domain interactions, which are currently poorly understood. We have used NMR to study structures and dynamics of NRPS single- and multi-domains. We show that the domains are neither subject to random interactions nor do they display a fixed quaternary organization. Instead, NRPSs are subject to multiple transient domain interactions which are accompanied by a modulation of their internal dynamics. Understanding the dynamic mechanism of NRPS domain communication may open the venue to efficient NRPS assembly line reprogramming and the production of novel pharmaceuticals.

Transient Domain Interactions in Non-Ribosomal Peptide Synthetases

Non-ribosomal peptide synthetases (NRPSs) are multi-module, multi-domain enzymes, synthesizing a wealth of important natural products in bacteria and fungi. These products often find pharmaceutical applications as antibiotics, antitumor agents or immunosuppressant drugs. NRPS use a remarkable assembly line organization to covalently load substrates into each module and catalyze peptide bond formation between substrates loaded on adjacent modules. The substrates may optionally be modified by tailoring domains. These multiple catalytic steps require a series of sequential domain interactions, which are currently poorly understood. We have used NMR to study structures and dynamics of NRPS single- and multi-domains. We show that the domains are neither subject to random interactions nor do they display a fixed quaternary organization. Instead, NRPSs are subject to multiple transient domain interactions which are accompanied by a modulation of their internal dynamics. Understanding the dynamic mechanism of NRPS domain communication may open the venue to efficient NRPS assembly line reprogramming and the production of novel pharmaceuticals.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-Site of the Peptidyl Transferase Center

The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a critical role.

Solubility Enhancement and Antibacterial Activity of Chloramphenicol Includedin Modified β-Cyclodextrins

CHL is widely used for the treatment of super-ficial eye infections and, because of its low cost and recognized efficacy, it is one of the most popular antibiotics in developing countries. Its mechanism of action has been elucidated and found to rely on the inhibition of peptidyl transferase, an RNA enzyme that catalyzes peptide bond formation between adjacent amino acids during protein synthesis.

The Roles of RNA in the Synthesis of Protein

The crystal structures of ribosomes that have been obtained since 2000 have transformed our understanding of protein synthesis. In addition to proving that RNA is responsible for catalyzing peptide bond formation, these structures have provided important insights into the mechanistic details of how the ribosome functions. This review emphasizes what has been learned about the mechanism of peptide bond formation, the antibiotics that inhibit ribosome function, and the fidelity of decoding.

Validating a new proton shuttle reaction pathway for formation of the peptide bond in ribosomes: A theoretical investigation

A ribosome is a large RNA–protein machine that catalyzes protein synthesis. RNA plays a crucial role in catalyzing peptide bond formation. In this Letter, we focus on the catalytic role of the A76 2′-OH of the P site in tRNA. Three possible reaction schemes were compared using the same reaction model and theoretical level. By comparison of the potential energy surface and optimized geometries of transition states, a new favorable reaction pathway is shown. The activation energy is 24 kcal/mol. This work will provide a foundation for further theoretical and experimental study of ribosome mechanisms.

The ribosome catalyzes peptide bond formation by providing high ionic strength

We performed QM/MM simulations based on density functional theory (DFT) and the density-functional tight binding method (DFTB) to investigate the reaction mechanism of the peptide-bond formation in the ribosome in atomistic detail. We found the key role of the ribosome in the increased availability of mobile ions, the counter-ions to the negatively charged RNA. To form the peptide bond, a C–N bond between the two amino acids is formed, a C–O bond between one amino acid and tRNA is broken, and a hydrogen atom is transferred from the N to the O atom. We found the hydrogen transfer to occur in two mechanisms in a competing manner with similar activation energies: a direct transfer, and a proton-shuttle mechanism via a ribose-2′-OH group. For this system it was found to be vital to calculate the energy barrier in numerous snapshots taken from molecular dynamics simulations and average them. Advantages and disadvantages of an exponential average compared with a direct average between the snapshots are discussed. An energy decomposition of the QM/MM results shows that the catalytic function is caused by the electrostatic influence of the environment rather than by mere positioning of the reactants. Analysis of the electrostatic influence residue-by-residue showed the importance of (sodium) ions near the active site. The free energy of activation for the direct proton-transfer mechanism was calculated by umbrella sampling. It confirmed a moderate entropic contribution to the activation free energy found in experiment. Overall, this study increases our understanding of the catalytic mechanism of the ribosome and probably also other ribozymes.

Engineered transaminopeptidase, aminolysin-S for catalysis of peptide bond formation to give linear and cyclic dipeptides by one-pot reaction

Aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 was engineered into transaminopeptidase and used to catalyze an aminolysis reaction to give linear and cyclic dipeptides from cost-effective substrates such as the ester derivatives of amino acids.

The evolving ribosome: from non-coded peptide bond formation to sophisticated translation machinery

Structural analysis supported by biochemical, mutagenesis and computational evidence, revealed that the contemporary ribosome's active site is a universal symmetrical pocket made of ribosomal RNA. This pocket seems to be the remnant of the proto-ribosome, a dimeric RNA assembly evolved by gene duplication, capable of autonomously catalyzing peptide bond formation and non-coded amino acid polymerization.

Identification of the prebiotic translation apparatus within the contemporary ribosome

AbstractA structural element that could have existed independently in the prebiotic era was identified at the active site of the contemporary ribosome. It is suggested to have functioned as a proto-ribosome catalyzing peptide bond formation and non-coded elongation in the same manner that contemporary ribosomes exert positional catalysis, namely by accommodating the reactants in stereochemistry favourable for inline nucleophilic attack. This simple apparatus is a dimer of self-folding RNA units that could have assembled spontaneously into a symmetrical pocket-like structure, sufficiently efficient to be preserved throughout evolution as the active site of modern ribosomes, thus presenting a conceivable starting point for translation.Here we discuss the proto-ribosome emergence hypothesis and show that the tendency for dimerization, a prerequisite for obtaining the catalytic centre, is linked to the fold of its two components, indicating functional selection at the molecular level in the prebiotic era and supporting the existence of dimeric proto-ribosome.

Synthesis of a Fluorine-Substituted Puromycin Derivative for Brønsted Studies of Ribosomal-Catalyzed Peptide Bond Formation

The mechanism by which the ribosome catalyzes peptide bond formation remains controversial. Here we describe the synthesis of a nucleoside that can be used in Brønsted experiments to assess the transition state of ribosome catalyzed peptide bond formation. This substrate is the nucleoside 3'-amino-3'-deoxy-3'-[(3''R)-3-fluoro-l-phenyl-alanyl]-N(6),N(6)-dimethyladenosine, which was prepared from (1R,2R)-2-amino-1-phenylpropane-1,3-diol. This substrate is active in peptide bond formation on the ribosome and is a useful probe for Brønsted analysis experiments on the ribosome.

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp >> Pro, with Pro being by far the slowest. However, when Phe-tRNA(Phe) was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was approximately 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.

Irreversible chemical steps control intersubunit dynamics during translation

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

Structural and functional prerequisites for ribosomal nascentpeptide acceptors: Attempts to decipher the nature of the ribosome's catalysis of peptide bond formation.

Aminoacyl ribonucleoside analogues that are capable of binding to the acceptor site of ribosomes and taking over the nascent peptide bear, if properly designed, the potential of antibiotic and cytostatic activity. Here we present a study on the intrinsic conformations of natural and synthetic peptide acceptors and the basicities of their peptide accepting amino groups. The conformations and thermodynamic parameters of several synthetic puromycin analogues have been elucidated through ab initio calculations as well as temperature and pH dependent (1)H NMR experiments. The intrinsic basicities of their peptide accepting amino groups were determined through (1)H NMR and compared to the effective basicities of the peptide accepting amino groups of aminoacyl transfer RNAs with the same amino acid side chains, as estimated from the pH dependent kinetics of mRNA programmed ribosomal peptidyl transfer.

The Kinetics of Ribosomal Peptidyl Transfer Revisited

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.

An Uncharged Amine in the Transition State of the Ribosomal Peptidyl Transfer Reaction

The ribosome has an active site comprised of RNA that catalyzes peptide bond formation. To understand how RNA promotes this reaction requires a detailed understanding of the chemical transition state. Here, we report the Brønsted coefficient of the alpha-amino nucleophile with a series of puromycin derivatives. Both 50S subunit- and 70S ribosome-catalyzed reactions displayed linear free-energy relationships with slopes close to zero under conditions where chemistry is rate limiting. These results indicate that, at the transition state, the nucleophile is neutral in the ribosome-catalyzed reaction, in contrast to the substantial positive charge reported for typical uncatalyzed aminolysis reactions. This suggests that the ribosomal transition state involves deprotonation to a degree commensurate with nitrogen-carbon bond formation. Such a transition state is significantly different from that of uncatalyzed aminolysis reactions in solution.