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• W2117325595 abstract "Peptide bond formation on the ribosome takes place in an active site composed of RNA. Recent progress of structural, biochemical, and computational approaches has provided a fairly detailed picture of the catalytic mechanism of the reaction. The ribosome accelerates peptide bond formation by lowering the activation entropy of the reaction due to positioning the two substrates, ordering water in the active site, and providing an electrostatic network that stabilizes the reaction intermediates. Proton transfer during the reaction appears to be promoted by a concerted proton shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate. Peptide bond formation on the ribosome takes place in an active site composed of RNA. Recent progress of structural, biochemical, and computational approaches has provided a fairly detailed picture of the catalytic mechanism of the reaction. The ribosome accelerates peptide bond formation by lowering the activation entropy of the reaction due to positioning the two substrates, ordering water in the active site, and providing an electrostatic network that stabilizes the reaction intermediates. Proton transfer during the reaction appears to be promoted by a concerted proton shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate. Protein synthesis in the cell is performed on ribosomes, large ribonucleoprotein particles that consist of three RNA molecules and more than 50 proteins. Ribosomes are composed of two subunits, the larger of which has a sedimentation coefficient of 50S in prokaryotes (the 50S subunit) and the smaller which sediments at 30S (the 30S subunit); together they form 70S ribosomes. The ribosome is a molecular machine that selects its substrates, aminoacyl-tRNAs (aa-tRNAs), rapidly and accurately and catalyzes the synthesis of peptides from amino acids. The 30S subunit contains the decoding site, where base-pairing interactions between the mRNA codon and the tRNA anticodon determine the selection of the cognate aa-tRNA. The large ribosomal subunit contains the site of catalysis—the peptidyl transferase (PT) center—which is responsible for making peptide bonds during protein elongation and for the hydrolysis of peptidyl-tRNA (pept-tRNA) during the termination of protein synthesis. The ribosome has three tRNA binding sites: A, P, and E sites (Figure 1). During the elongation cycle of protein synthesis, aa-tRNA is delivered to the A site of the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Following GTP hydrolysis and release from EF-Tu, aa-tRNA accommodates in the A site of the PT center and reacts with pept-tRNA bound to the P site, yielding deacylated tRNA in the P site and A site pept-tRNA that is extended by one amino acid residue. The subsequent movement of tRNAs and mRNA through the ribosome (translocation) is catalyzed by another elongation factor (EF-G in bacteria). During translocation, pept-tRNA and deacylated tRNA move to the P and E sites, respectively; a new codon is exposed in the A site for the interaction with the next aa-tRNA, and the deacylated tRNA is released from the E site. The reaction catalyzed by the PT center of the ribosome is the aminolysis of an ester bond, with the nucleophilic α-amino group of A site aa-tRNA attacking the carbonyl carbon of the ester bond linking the peptide moiety of P site pept-tRNA. The reactivity of esters with amines intrinsically is rather high, as the uncatalyzed reaction proceeds with a rate of ∼10−4 M−1s−1 at room temperature. The ribosome increases the rate of peptide bond formation by 106- to 107-fold, and it may catalyze the reaction in various ways, including chemical catalysis employing ribosome residues as general acids and bases, electrostatic stabilization of the zwitterionic transition state, desolvation, or simply by bringing the two substrates into close proximity and correct orientation to each other. In the last few years, a fairly consistent picture of the catalytic mechanism of peptide bond formation on the ribosome has emerged due to the progress in ribosome crystallography, as well as kinetic, biochemical, genetic, and computational approaches. In this review, we summarize these recent results and present a current model for the mechanism of peptide bond formation on the ribosome. The investigation of peptide bond formation started when Monro and colleagues showed that the PT active site is located on the large ribosomal subunit (Monro, 1967Monro R.E. Catalysis of peptide bond formation by 50 S ribosomal subunits from Escherichia coli.J. Mol. Biol. 1967; 26: 147-151Crossref PubMed Scopus (143) Google Scholar, Monro and Marcker, 1967Monro R.E. Marcker K.A. Ribosome-catalysed reaction of puromycin with a formylmethionine-containing oligonucleotide.J. Mol. Biol. 1967; 25: 347-350Crossref PubMed Scopus (155) Google Scholar). Those studies were performed with the so-called “fragment reaction” utilizing low-molecular-weight compounds as substrate analogs. An N-blocked aminoacylated oligonucleotide, such as CCA-fMet, and puromycin (Pmn; O-methyl tyrosine linked to N6-dimethyl adenosine via an amide bond) were used as substrates mimicking the aminoacylated terminus of tRNAs in the P and A sites, respectively. Unfortunately, the fragment reaction required high concentrations of alcohol, and the observed reaction rates appeared to be much slower than the rates of protein synthesis in vivo, raising the question of whether such a decidedly nonphysiological system was representative for the reaction in the cell. In particular, the possibility remained that the presence of the small subunit was necessary to induce a conformation of the active site favoring the reaction (Bashan et al., 2003Bashan A. Agmon I. Zarivach R. Schluenzen F. Harms J. Berisio R. Bartels H. Franceschi F. Auerbach T. Hansen H.A. et al.Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.Mol. Cell. 2003; 11: 91-102Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). One of the problems of the fragment reaction was the low affinity of small substrate analogs to their binding sites on the 50S subunit. To solve this problem, Steitz, Strobel, and colleagues designed new, somewhat larger substrate analogs that could be used in a modified, alcohol-free version of the fragment reaction (Schmeing et al., 2002Schmeing T.M. Seila A.C. Hansen J.L. Freeborn B. Soukup J.K. Scaringe S.A. Strobel S.A. Moore P.B. Steitz T.A. A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits.Nat. Struct. Biol. 2002; 9: 225-230PubMed Google Scholar, Schmeing et al., 2005aSchmeing T.M. Huang K.S. Kitchen D.E. Strobel S.A. Steitz T.A. Structural insights into the roles of water and the 2′ hydroxyl of the P site tRNA in the peptidyl transferase reaction.Mol. Cell. 2005; 20: 437-448Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Schmeing et al., 2005bSchmeing T.M. Huang K.S. Strobel S.A. Steitz T.A. An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA.Nature. 2005; 438: 520-524Crossref PubMed Scopus (265) Google Scholar). Furthermore, when full-length pept-tRNA and Pmn or cytidine-Pmn (CPmn) were used, the reaction rates on isolated 50S subunits were comparable to those measured on the 70S ribosomes (Wohlgemuth et al., 2006Wohlgemuth I. Beringer M. Rodnina M.V. Rapid peptide bond formation on isolated 50S ribosomal subunits.EMBO Rep. 2006; 7: 669-703Crossref Scopus (36) Google Scholar) or observed in vivo. This strongly supports the conclusion that the 50S subunit alone possesses the full potential of catalyzing peptide bond formation (Wohlgemuth et al., 2006Wohlgemuth I. Beringer M. Rodnina M.V. Rapid peptide bond formation on isolated 50S ribosomal subunits.EMBO Rep. 2006; 7: 669-703Crossref Scopus (36) Google Scholar). The elucidation of the catalytic mechanism of peptide bond formation required a complete reconstituted in vitro translation system in which parameters such as pH, temperature, and ionic conditions could be changed. One critical issue in such experiments is to show that the rate of product formation reflects the chemistry step, rather than substrate binding or conformational rearrangements. In a complete translation system, this condition is unlikely to be fulfilled, because the overall rate of protein synthesis is limited by aa-tRNA selection, which ensures that only the correct amino acid will be incorporated into the protein. The movement of aa-tRNA into the A site is a multistep process that requires structural rearrangements of the ribosome, EF-Tu, and aa-tRNA (reviewed in Rodnina et al., 2005bRodnina M.V. Gromadski K.B. Kothe U. Wieden H.J. Recognition and selection of tRNA in translation.FEBS Lett. 2005; 579: 938-942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Binding of aa-tRNA in complex with EF-Tu·GTP to the ribosome and codon recognition results in GTP hydrolysis by EF-Tu. Aa-tRNA is released from EF-Tu·GDP and moves through the ribosome into the PT center where its 3′-terminal aminoacylated CCA end is engaged in multiple interactions with the rRNA (Kim and Green, 1999Kim D.F. Green R. Base-pairing between 23S rRNA and tRNA in the ribosomal A site.Mol. Cell. 1999; 4: 859-864Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, Yusupov et al., 2001Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T.N. Cate J.H. Noller H.F. Crystal structure of the ribosome at 5.5 Å resolution.Science. 2001; 292: 883-896Crossref PubMed Scopus (1612) Google Scholar, Hansen et al., 2002Hansen J.L. Schmeing T.M. Moore P.B. Steitz T.A. Structural insights into peptide bond formation.Proc. Natl. Acad. Sci. USA. 2002; 99: 11670-11675Crossref PubMed Scopus (230) Google Scholar, Bashan et al., 2003Bashan A. Agmon I. Zarivach R. Schluenzen F. Harms J. Berisio R. Bartels H. Franceschi F. Auerbach T. Hansen H.A. et al.Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.Mol. Cell. 2003; 11: 91-102Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The rate of accommodation of aa-tRNA in the A site is ∼10 s−1, and peptide bond formation follows instantaneously (Pape et al., 1998Pape T. Wintermeyer W. Rodnina M.V. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.EMBO J. 1998; 17: 7490-7497Crossref PubMed Scopus (306) Google Scholar, Bieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google Scholar). Because accommodation precedes peptide bond formation, it limits the rate of product formation as long as it is slower than peptidyl transfer (Pape et al., 1998Pape T. Wintermeyer W. Rodnina M.V. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.EMBO J. 1998; 17: 7490-7497Crossref PubMed Scopus (306) Google Scholar). Thus, studying the catalytic mechanism of peptide bond formation is possible only when peptidyl transfer is uncoupled from accommodation. One possibility to overcome the accommodation problem is to decrease the rate of the chemistry reaction such that it becomes significantly slower than the tRNA accommodation step and thus amenable for biochemical analysis. This was accomplished by replacing the reactive nucleophilic α-NH2 group by the much less reactive OH group (Bieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google Scholar). The OH derivatives bind to the PT center in the same way as unmodified substrates (Schmeing et al., 2005bSchmeing T.M. Huang K.S. Strobel S.A. Steitz T.A. An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA.Nature. 2005; 438: 520-524Crossref PubMed Scopus (265) Google Scholar), and the reaction with Pmn-OH exhibited the same activation enthalpy as with unmodified Pmn (Table 1), suggesting that the change of the nucleophilic group did not considerably alter the reaction pathway. The hydroxyl derivative of Phe-tRNA (OH-tRNA) was fully reactive with fMet-tRNA in the P site, but, because of the decreased nucleophilicity of the attacking group, the rate of reaction was much lower, 10−3 s−1, i.e., far below the rate of accommodation.Table 1Activation Parameters of Uncatalyzed and Catalyzed Peptide Bond FormationAmineEsterRateActivation Parameters (kcal/mol)ΔG≠ΔH≠TΔS≠UncatalyzedTrisfGly-ethylene glycol10−4 (M−1s−1)22.29.1−13.1TrisfMet-tRNAfMet10−4 (M−1s−1)22.716.2−6.5Catalyzed by 70S RibosomesPmnaMeasured at limiting concentrations of Pmn; the rate obtained at these conditions (kcat/KM) is comparable to the second-order reaction of model substrates in solution (25°C) (Sievers et al., 2004).fMetPhe-tRNAPhe103 (M−1s−1)14.016.02.0PmnbMeasured at saturating concentration of Pmn or OH-Pmn (kcat conditions) (25°C) (Rodnina et al., 2005a).fMetPhe-tRNAPhe5 (s−1)16.517.20.7OH-PmnbMeasured at saturating concentration of Pmn or OH-Pmn (kcat conditions) (25°C) (Rodnina et al., 2005a).fMet-tRNAfMet6 × 10−3 (s−1)20.516.8−3.7fGly, N-formyl glycine; fMet, N-formyl methionine.a Measured at limiting concentrations of Pmn; the rate obtained at these conditions (kcat/KM) is comparable to the second-order reaction of model substrates in solution (25°C) (Sievers et al., 2004Sievers A. Beringer M. Rodnina M.V. Wolfenden R. The ribosome as an entropy trap.Proc. Natl. Acad. Sci. USA. 2004; 101: 7897-7901Crossref PubMed Scopus (232) Google Scholar).b Measured at saturating concentration of Pmn or OH-Pmn (kcat conditions) (25°C) (Rodnina et al., 2005aRodnina M.V. Beringer M. Bieling P. Ten remarks on peptide bond formation on the ribosome.Biochem. Soc. Trans. 2005; 33: 493-498Crossref PubMed Scopus (16) Google Scholar). Open table in a new tab fGly, N-formyl glycine; fMet, N-formyl methionine. For general acid-base catalysis to occur in an aqueous environment at physiological conditions, the pKa values of the catalytic groups have to be close to neutrality in order to efficiently abstract or donate a proton during the reaction. Measuring the reaction rate at different pH values in the range between 6 and 9 revealed that the rate of the reaction between hydroxy-Phe-tRNAPhe and fMet-tRNA was not affected by pH changes (Table 2). This result indicates that catalysis by the PT center intrinsically is independent of pH and argues against an involvement of ionizing groups of the ribosome in the catalytic mechanism of the PT reaction (Bieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google Scholar). Furthermore, peptide bond formation between full-length pept-tRNA and unmodified aa-tRNAs did not show any pH dependence (Bieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google Scholar), and—although the rate-limiting accommodation step probably would have masked part of a potential pH effect—these results are consistent with a small, if any, influence of ionizing groups of the ribosome on the reaction. Taking into account the ionization of the α-NH2 group of aa-tRNA (pKa = 8) and a measured reaction rate of 6 s−1 observed at pH 6, the maximum rate of peptidyl transfer with unmodified full-length tRNA can be estimated to be intrinsically very high, >300 s−1 (Bieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google Scholar).Table 2Inhibition of Peptide Bond Formation by Protonation of Ribosomal GroupsA Site SubstrateRate DecreaseReferencePmn150-foldKatunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google ScholarOH-Pmn150-foldKatunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google ScholarC-PmnNoneBrunelle et al., 2006Brunelle J.L. Youngman E.M. Sharma D. Green R. The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity.RNA. 2006; 12: 33-39Crossref PubMed Scopus (75) Google Scholaraa-tRNANoneBieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google ScholarOH-tRNANoneBieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google Scholar Open table in a new tab Another possibility to separate accommodation and peptidyl transfer steps is to use substrate analogs that bind to the A site rapidly and do not require accommodation. In such experiments, 70S ribosomes programmed with natural mRNA and carrying initiator fMet-tRNA or pept-tRNA in the P site were reacted with Pmn as A site substrate analog (Katunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The reaction rate was not limited by Pmn binding (Katunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Sievers et al., 2004Sievers A. Beringer M. Rodnina M.V. Wolfenden R. The ribosome as an entropy trap.Proc. Natl. Acad. Sci. USA. 2004; 101: 7897-7901Crossref PubMed Scopus (232) Google Scholar), and the kinetics of the catalytic step could be monitored by quench flow. The maximum rate of peptide bond formation, measured with pept-tRNA as P site substrate, was about 50 s−1 at pH values >8 (Katunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Youngman et al., 2004Youngman E.M. Brunelle J.L. Kochaniak A.B. Green R. The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release.Cell. 2004; 117: 589-599Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The rate of reaction varied within a factor of 50–100 depending on the length of the peptidyl moiety of the P site tRNA, the C-terminal amino acid of the peptide, or the identity of the tRNA in the P site: the reaction rate was close to 1 s−1 with fMet-tRNAfMet in the P site, 10–20 s−1 with di- and tripeptidyl-tRNAs fMetPhe-tRNAPhe and fMetPhePhe-tRNAPhe (Katunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Brunelle et al., 2006Brunelle J.L. Youngman E.M. Sharma D. Green R. The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity.RNA. 2006; 12: 33-39Crossref PubMed Scopus (75) Google Scholar), and 50 s−1 with fMetAlaAsnMetPheAla-tRNAAla (Katunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In contrast to full-length aminoacyl-tRNA, pronounced pH dependence was observed for peptide bond formation with the minimal A site substrate, Pmn (Table 2). Protonation of a ribosomal group with a pKa around 7.5 reduced the rate of reaction about 150-fold (Katunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Brunelle et al., 2006Brunelle J.L. Youngman E.M. Sharma D. Green R. The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity.RNA. 2006; 12: 33-39Crossref PubMed Scopus (75) Google Scholar). This effect may in principle indicate an involvement of a ribosome residue as general base in reaction, although the magnitude of inhibition by protonation of a group with pKa of 7.5 is much less than expected for an essential base. Alternatively, the pH effect may reflect a conformational rearrangement of active-site residues that impairs catalysis and does not take place with full-length aa-tRNA as A site substrate (Katunin et al., 2002Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Beringer et al., 2005Beringer M. Bruell C. Xiong L. Pfister P. Bieling P. Katunin V.I. Mankin A.S. Bottger E.C. Rodnina M.V. Essential mechanisms in the catalysis of peptide bond formation on the ribosome.J. Biol. Chem. 2005; 280: 36065-36072Crossref PubMed Scopus (72) Google Scholar, Bieling et al., 2006Bieling P. Beringer M. Adio S. Rodnina M.V. Peptide bond formation does not involve acid-base catalysis by ribosomal residues.Nat. Struct. Mol. Biol. 2006; 13: 423-428Crossref PubMed Scopus (96) Google Scholar). This conclusion is corroborated by the observation that the reaction between C-Pmn in the A site and pept-tRNA in the P site was not influenced by the ionization of ribosomal groups (Brunelle et al., 2006Brunelle J.L. Youngman E.M. Sharma D. Green R. The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity.RNA. 2006; 12: 33-39Crossref PubMed Scopus (75) Google Scholar), suggesting that the presence of the cytidine residue, which mimics C75 of the A site tRNA and presumably its interaction with G2553, was sufficient to induce and stabilize the active conformation of the PT center. Generally, the reaction with full-length tRNA seems to be more robust than the reaction with Pmn, indicating the importance of more remote interactions for positioning the tRNA in the A site. Even when base pairs between C74 or C75 of P site tRNA with rRNA residues G2252 or G2251, respectively, were disrupted by mutagenesis, there was no effect on the rate of peptide bond formation with aa-tRNA as A site substrate (Feinberg and Joseph, 2006Feinberg J.S. Joseph S. A conserved base-pair between tRNA and 23S rRNA in the peptidyl transferase center is important for peptide release.J. Mol. Biol. 2006; 364: 1010-1020Crossref PubMed Scopus (20) Google Scholar). Crucial information about the mechanism of catalysis can be obtained by comparing the thermodynamic properties of the catalyzed and uncatalyzed reactions. Enzymes that employ general acid-base catalysis act by lowering the activation enthalpy of the reaction. If the ribosome acted as such a chemical catalyst, then the rate enhancement produced by the ribosome should result from a reduction in the enthalpy of activation. If, on the other hand, the ribosome used other mechanisms of catalysis, such as substrate positioning in the active site, desolvation, or electrostatic shielding, then the rate enhancement produced by the ribosome should be largely entropic in origin. Comparing the rate of the ribosome-catalyzed reaction with a second-order model reaction in solution revealed that the acceleration is achieved by a major lowering of the entropy of activation (Sievers et al., 2004Sievers A. Beringer M. Rodnina M.V. Wolfenden R. The ribosome as an entropy trap.Proc. Natl. Acad. Sci. USA. 2004; 101: 7897-7901Crossref PubMed Scopus (232) Google Scholar) (Table 1), whereas the enthalpy of activation was practically unchanged (Rodnina et al., 2005aRodnina M.V. Beringer M. Bieling P. Ten remarks on peptide bond formation on the ribosome.Biochem. Soc. Trans. 2005; 33: 493-498Crossref PubMed Scopus (16) Google Scholar). Consistent with the pH independence of the reaction with full-length tRNA, these findings suggest that general acid-base catalysis by ribosomal residues does not play a significant role in peptidyl transfer on the ribosome. Some details of the reaction pathway, and particularly the exact difference between the reactions on the ribosome and in solution, are still not understood. Measurements of kinetic isotope effects of the reaction on the 50S subunit (Seila et al., 2005Seila A.C. Okuda K. Nunez S. Seila A.F. Strobel S.A. Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction.Biochemistry. 2005; 44: 4018-4027Crossref PubMed Scopus (38) Google Scholar) suggested that the ribosome may promote peptide bond formation by a mechanism that differs in detail from the uncatalyzed aminolysis reaction in solution (Jencks and Gilchrist, 1968Jencks W.P. Gilchrist M. Nonlinear structure-reactivity correlations. The reactivity of nucleophilic reagents toward esters.J. Am. Chem. Soc. 1968; 90: 2622-2637Crossref Scopus (564) Google Scholar); the reaction characteristics on the 50S subunit are most consistent with the model in which the formation of the tetrahedral intermediate is the first irreversible step of the reaction and deprotonation of the amine is occurring concurrently with intermediate formation (Seila et al., 2005Seila A.C. Okuda K. Nunez S. Seila A.F. Strobel S.A. Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction.Biochemistry. 2005; 44: 4018-4027Crossref PubMed Scopus (38) Google Scholar). A very low Brønsted coefficient (∼0.2) suggests little charge development on the amine in the transition state (Seila et al., 2005Seila A.C. Okuda K. Nunez S. Seila A.F. Strobel S.A. Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction.Biochemistry. 2005; 44: 4018-4027Crossref PubMed Scopus (38) Google Scholar). On the other hand, molecular dynamics simulation suggested a late transition state for C-N bond formation and strong H bonding between the nucleophile and the P site O2′ before the proton is transferred, which implies that the geometry and charge distribution of the rate-limiting transition state may be similar to that of the high energy intermediate, irrespective of whether it actually is formed before or after the intermediate (Trobro and Åqvist, 2006Trobro S. Åqvist J. Analysis of predictions for the catalytic mechanism of ribosomal peptidyl transfer.Biochemistry. 2006; 45: 7049-7056Crossref PubMed Scopus (77) Google Scholar). 50S subunits are composed of two rRNA molecules, 23S rRNA and 5S rRNA, and more than 30 proteins (Figure 2A). Based on crosslinking studies, the PT center was identified in domain V of 23S rRNA with its interacting proteins (Noller, 1991Noller H.F. Ribosomal RNA and translation.Annu. Rev. Biochem. 1991; 60: 191-227Crossref PubMed Scopus (393) Google Scholar). Biochemical studies showed that 50S subunits largely depleted of proteins retained PT activity (Noller et al., 1992Noller H.F. Hoffarth V. Zimniak L. Unusual resistance of peptidyl transferase to protein extraction procedures.Science. 1992; 256: 1416-1419Crossref PubMed Scopus (553) Google Scholar). High-resolution crystal structures of the 50S subunit from Haloarcula marismortui revealed that the PT center is composed of RNA only, with no protein within 15 Å of the active site (Ban et al., 2000Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution.Science. 2000; 289: 905-920Crossref PubMed Scopus (2709) Google Scholar). Crystal structures of 50S subunits from Deinococcus radiodurans (Harms et al., 2001Harms J. Schluenzen F. Zarivach R. Bashan A. Gat S. Agmon I. Bartels H. Franceschi F. Yonath A. High resolution structure of the large ribosomal subunit from a mesophilic eubacterium.Cell. 2001; 107: 679-688Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, Bashan et al., 2003Bashan A. Agmon I. Zarivach R. Schluenzen F. Harms J. Berisio R. Bartels H. Franceschi F. Auerbach T. Hansen H.A. et al.Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.Mol. Cell. 2003; 11: 91-102Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar" @default.
• W2117325595 created "2016-06-24" @default.
• W2117325595 creator A5028476493 @default.
• W2117325595 creator A5035464151 @default.
• W2117325595 date "2007-05-01" @default.
• W2117325595 modified "2023-10-12" @default.
• W2117325595 title "The Ribosomal Peptidyl Transferase" @default.
• W2117325595 cites W1438574067 @default.
• W2117325595 cites W1575818733 @default.
• W2117325595 cites W1901863113 @default.
• W2117325595 cites W1969681263 @default.
• W2117325595 cites W1973776965 @default.
• W2117325595 cites W1975455557 @default.
• W2117325595 cites W1983534198 @default.
• W2117325595 cites W1983948144 @default.
• W2117325595 cites W1984884697 @default.
• W2117325595 cites W1989794771 @default.
• W2117325595 cites W1993667465 @default.
• W2117325595 cites W1996613940 @default.
• W2117325595 cites W1999362400 @default.
• W2117325595 cites W2000699855 @default.
• W2117325595 cites W2001560423 @default.
• W2117325595 cites W2001916877 @default.
• W2117325595 cites W2003433740 @default.
• W2117325595 cites W2010000945 @default.
• W2117325595 cites W2012419095 @default.
• W2117325595 cites W2015041612 @default.
• W2117325595 cites W2020692930 @default.
• W2117325595 cites W2020942151 @default.
• W2117325595 cites W2022754910 @default.
• W2117325595 cites W2029026643 @default.
• W2117325595 cites W2033929454 @default.
• W2117325595 cites W2037024999 @default.
• W2117325595 cites W2039951296 @default.
• W2117325595 cites W2044133901 @default.
• W2117325595 cites W2047706687 @default.
• W2117325595 cites W2049943436 @default.
• W2117325595 cites W2056093578 @default.
• W2117325595 cites W2057733356 @default.
• W2117325595 cites W2060467605 @default.
• W2117325595 cites W2073149987 @default.
• W2117325595 cites W2080354279 @default.
• W2117325595 cites W2086765626 @default.
• W2117325595 cites W2087439656 @default.
• W2117325595 cites W2087679230 @default.
• W2117325595 cites W2089457247 @default.
• W2117325595 cites W2092212918 @default.
• W2117325595 cites W2093041571 @default.
• W2117325595 cites W2093091879 @default.
• W2117325595 cites W2093471887 @default.
• W2117325595 cites W2097654784 @default.
• W2117325595 cites W2099155786 @default.
• W2117325595 cites W2101114936 @default.
• W2117325595 cites W2110557219 @default.
• W2117325595 cites W2114833790 @default.
• W2117325595 cites W2118391959 @default.
• W2117325595 cites W2133933397 @default.
• W2117325595 cites W2138341129 @default.
• W2117325595 cites W2145440568 @default.
• W2117325595 cites W2150503741 @default.
• W2117325595 cites W2154939230 @default.
• W2117325595 cites W2155373804 @default.
• W2117325595 cites W2156428769 @default.
• W2117325595 cites W2157013651 @default.
• W2117325595 cites W2169751581 @default.
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