The syn-Oriented 2-OH Provides a Favorable Proton Transfer Geometry in 1,2-Diol Monoester Aminolysis:  Implications for the Ribosome Mechanism

Rangelov, Miroslav; Vayssilov, Georgi N.; Yomtova, Vihra M.; Petkov, Dimiter D.

doi:10.1021/ja060648x

Cited by 47 publications

(53 citation statements)

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“…It is important to note that the ribosome does not provide groups that act as general acids or bases 2,[21][22][23] . The proton shuttles as depicted result in the protonation of the carbonyl oxygen before the less favourable protonation of the leaving 39-oxygen, following previous proposals 24,25 . The catalysis of peptide bond formation on the ribosome, compared with a spontaneous model reaction in solution, is entirely due to a change of the activation entropy, DS { (ref.…”

Section: Research Lettersupporting

confidence: 72%

Different substrate-dependent transition states in the active site of the ribosome

Kuhlenkoetter

Wintermeyer

Rodnina

2011

Nature

127

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The active site of the ribosome, the peptidyl transferase centre, catalyses two reactions, namely, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release-factordependent hydrolysis of peptidyl-tRNA. Unlike peptide bond formation, peptide release is strongly impaired by mutations of nucleotides within the active site, in particular by base exchanges at position A2602 (refs 1, 2). The 29-OH group of A76 of the peptidyl-tRNA substrate seems to have a key role in peptide release 3 . According to computational analysis 4 , the 29-OH may take part in a concerted 'proton shuttle' by which the leaving group is protonated, in analogy to similar current models of peptide bond formation 4-6 . Here we report kinetic solvent isotope effects and proton inventories (reaction rates measured in buffers with increasing content of deuterated water, D 2 O) of the two reactions catalysed by the active site of the Escherichia coli ribosome. The transition state of the release factor 2 (RF2)-dependent hydrolysis reaction is characterized by the rate-limiting formation of a single strong hydrogen bond. This finding argues against a concerted proton shuttle in the transition state of the hydrolysis reaction. In comparison, the proton inventory for peptide bond formation indicates the rate-limiting formation of three hydrogen bonds with about equal contributions, consistent with a concerted eightmembered proton shuttle in the transition state 5 . Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase centre.Peptide bond formation and peptide release involve the attack of different nucleophilic groups on the ester carbonyl carbon of peptidyltransfer (t)RNA in the P site of the ribosome; these nucleophilic groups are respectively the a-amino group of A-site-bound aminoacyl-tRNA and a water molecule. Unlike peptide bond formation, which does not require auxiliary factors, peptide release is assisted by termination (release) factors; in E. coli, these are RF1 and RF2. On recognition of a stop codon in the decoding site, the conserved GGQ motif of RF1/2 is inserted into the peptidyl transferase centre, augmenting the active site and inducing the hydrolysis of peptidyl-tRNA (see ref. 3 for a review). Release-factor binding induces a conformational change that involves conserved 23S ribosomal RNA residues, in particular U2506 and U2585 (refs 6-10), and opens the active site for the access of water 11 . The conserved Gln residue in RF1/2 has been attributed an essential function in positioning the hydrolytic water molecule and accounts for the high specificity of the active site for water as the nucleophile, discriminating against a larger amine 12 . Computational analysis suggested that the 29-OH of A76 of the P-site tRNA may take part in a concerted proton shuttle mechanism for the protonation of the leaving 39-O (ref. 4), in analogy to the mechanism suggested for peptide bond formation 5,13 . A fully concerted proton shuttle would ...

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Section: Research Lettersupporting

confidence: 72%

Different substrate-dependent transition states in the active site of the ribosome

Kuhlenkoetter

Wintermeyer

Rodnina

2011

Nature

127

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confidence: 97%

“…The presence of this water molecule had been predicted by molecular dynamics (MD) simulations (15, 17) and was later confirmed in a highresolution crystal structure of the RAP TS analog bound to the PTC (1). None of the other proposed mechanisms (26,27) have yielded reasonable energetics in calculations (15,28).Third, there is another water molecule that is in a perfect position to stabilize any developing negative charge in the transition state on the ester carbonyl oxygen of the P-site substrate. Simulations have predicted this water molecule to be hydrogen bonded to the carbonyl oxygen (15, 17), and it was subsequently observed experimentally (1,29).…”

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confidence: 99%

The transition state for peptide bond formation reveals the ribosome as a water trap

Wallin

Åqvist

2010

Proc. Natl. Acad. Sci. U.S.A.

103

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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...

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“…The structures of the Hma50 complexed with a peptidyl-CCA substrate or with an analogue of the intermediate show that the CCA bound in the P-site interacts with the P-loop, nucleotides 2246-2258 of the 23S rRNA, and that the attacking ␣-NH 2 group of a bound CC-puromycin substrate analogue is hydrogen-bonded to both the 2Ј-OH of the terminal A76 of the peptidyl-CCA P-site substrate and to the N3 of the ribosomal base A2451 (Escherichia coli numbering). The 2Ј-OH is essential for catalysis (8,(10)(11)(12)(13)(14)(15)(16)(17)(18) and is thought to provide a proton shuttle from the attacking ␣-NH 2 group to the P-site 3Ј-OH of A76, whereas A2451 assists in the proper positioning of the attacking ␣-NH 2 group. Currently, almost all structural, biochemical, genetic, and kinetic data are consistent with the proton shuttle mechanism (19) suggested initially by Dorner et al (13).…”

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confidence: 99%

Cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70S ribosome and the 50S subunit

Simonović

Steitz

2008

Proc. Natl. Acad. Sci. U.S.A.

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Recently, two crystal structures of the Thermus thermophilus 70S ribosome in the same functional state were determined at 2.8 and 3.7 Å resolution but were different throughout. The most functionally significant structural differences are in the conformation of the peptidyl-transferase center (PTC) and the interface between the PTC and the CCA end of the P-site tRNA. Likewise, the 3.7 Å PTC differed from the functionally equivalent structure of the Haloarcula marismortui 50S subunit. To ascertain whether the 3.7 Å model does indeed differ from the other two, we performed cross-crystal averaging of the two 70S data sets. The unbiased maps suggest that the conformation of the PTC-CCA in the two 70S crystal forms is identical to that of the 2.8 Å 70S model as well as that of the H. marismortui 50S subunit. We conclude that the structure of the PTC is the same in the functionally equivalent 70S ribosome and the 50S subunit.T he ribosome catalyzes the final step in the flow of genetic information from DNA to proteins using the decoding machinery situated in the small ribosomal subunit and the peptidyl-transferase center (PTC) located in the large ribosomal subunit. Insights into the structural basis of mRNA decoding have come largely from structures of the Thermus thermophilus 30S ribosomal subunit and its substrate complexes (1-4), whereas understanding of the structural basis of peptide bond formation has been derived from structures of the Haloarcula marismortui 50S subunit (Hma50) complexed with substrate, intermediate, and product analogues (5-9). The structures of the Hma50 complexed with a peptidyl-CCA substrate or with an analogue of the intermediate show that the CCA bound in the P-site interacts with the P-loop, nucleotides 2246-2258 of the 23S rRNA, and that the attacking ␣-NH 2 group of a bound CC-puromycin substrate analogue is hydrogen-bonded to both the 2Ј-OH of the terminal A76 of the peptidyl-CCA P-site substrate and to the N3 of the ribosomal base A2451 (Escherichia coli numbering). The 2Ј-OH is essential for catalysis (8,(10)(11)(12)(13)(14)(15)(16)(17)(18) and is thought to provide a proton shuttle from the attacking ␣-NH 2 group to the P-site 3Ј-OH of A76, whereas A2451 assists in the proper positioning of the attacking ␣-NH 2 group. Currently, almost all structural, biochemical, genetic, and kinetic data are consistent with the proton shuttle mechanism (19) suggested initially by Dorner et al. (13).Two crystal structures of the 70S ribosome from T. thermophilus (Tth70) at 2.8 and 3.7 Å resolution have recently been published. They represent the same functional state of the 70S particle with the P-and E-sites fully occupied albeit with different tRNAs (20, 21). The P-site is occupied by tRNA Phe in the 3.7 Å crystal and by tRNA fMet in the 2.8 Å crystal. The A-site is unoccupied in the 3.7 Å crystal but is partially occupied in the 2.8 Å crystal, with only the anticodon stem-loop region of tRNA Phe ordered and the antibiotic paromomycin bound to the decoding A-site. Moreover, whereas an endogenou...

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The syn-Oriented 2-OH Provides a Favorable Proton Transfer Geometry in 1,2-Diol Monoester Aminolysis: Implications for the Ribosome Mechanism

Cited by 47 publications

References 11 publications

Different substrate-dependent transition states in the active site of the ribosome

Different substrate-dependent transition states in the active site of the ribosome

The transition state for peptide bond formation reveals the ribosome as a water trap

Cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70S ribosome and the 50S subunit

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