Transition State Chirality and Role of the Vicinal Hydroxyl in the Ribosomal Peptidyl Transferase Reaction

Huang, Kevin S.; Carrasco, Nicolas; Pfund, Emmanuel; Strobel, Scott A.

doi:10.1021/bi800299u

Cited by 15 publications

(21 citation statements)

References 43 publications

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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: 70%

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: 70%

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

Kuhlenkoetter

Wintermeyer

Rodnina

2011

Nature

127

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“…Because of the proximity of the O2’ hydroxyl to the O3’ atom of A76 that is linked to the peptide chain, it was proposed that O2’ hydroxyl is critical for proton shuttling from the attacking α-amino group to the leaving O3’ hydroxyl. Indeed, it has been shown that the O2’ hydroxyl group remains neutral in the transition state confirming its role in the proton shuttle [73]. More recently, Strobel and colleagues have concluded that at the transition state the α-amino nucleophile is neutral, which is in a stark contrast to the development of the positively charged amino nucleophile during an uncatalyzed aminolysis reaction in solution [74].…”

Section: Catalytic Mechanismmentioning

confidence: 99%

A structural view on the mechanism of the ribosome-catalyzed peptide bond formation

Simonović

Steitz

2009

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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The ribosome is a large ribonucleoprotein particle that translates zgenetic 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.The ribosome is a large RNA-protein machine that catalyzes protein synthesis in all living organisms. It is composed of two subunits in all kingdoms of life. In eubacteria and archaea the large subunit comprises ~3,000 nucleotides of ribosomal RNA (rRNA), > 30 proteins and sediments at 50S, whereas the ~1,500 nucleotides and > 20 proteins of the smaller subunit sediment at 30S. These conveniently different sedimentation coefficients are used as descriptors of the large (50S) and the small (30S) ribosomal subunits, while the entire ribosomal particle is referred to as the 70S ribosome (in eukaryotes these are 60S, 40S and 80S, respectively). Not only do the two subunits differ in their size and morphology, as evidenced by the early micrographs of the entire E. coli 70S particle [1], but they also perform distinct roles during protein synthesis [2]. The 50S subunit contains the peptidyl-transferase center (PTC) that catalyzes the synthesis of peptide bond, whereas the 30S subunit contains the decoding center that ensures that the tRNA with the correct anticodon is bound to the ribosome and paired with the mRNA codon.

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“…This is important, as the 2 ¢ OH group can receive a proton only if it simultaneously donates a proton to some other group. However, model studies suggested that the carbonyl oxygen of the pept-tRNA, rather than the 3 ¢ OH, accepts the proton from the 2 ¢ OH (Huang et al 2008 ;Rangelov et al 2006 ) . Such a shuttle mechanism may be six-membered, with two protons simultaneously changing their positions in the TS, or eight-membered with three protons "in fl ight" in the TS; the latter TS includes a water molecule which is found in the right position in the crystal structures of the 50S subunits in the complex with TS analogs (Schmeing et al 2005a ;Wallin and Aqvist 2010 ) .…”

Section: Peptide Bond Formationmentioning

confidence: 99%

Rapid Kinetic Analysis of Protein Synthesis

Rodnina

Wintermeyer

2012

Biophysical Approaches to Translational Control of Gene Expression

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Protein synthesis comprises a sequence of consecutive reactions that entail recognition of substrates by the ribosome, synthesis of chemical bonds, and release of the reaction products. Each of the translation phases-initiation, elongation, termination, and ribosome recycling-is itself a multistep process. To achieve the overall high rate of protein synthesis, with ribosomes initiating roughly every 3 s on an mRNA and about 0.1 s required for each cycle of nascent peptide elongation, each of the individual reactions on the ribosome must be rapid. This implies that the reaction intermediates of protein synthesis are short-lived, with lifetimes in the few milliseconds range. The overall process is strongly biased towards forward reactions due to the combination of dynamic fl uctuations with irreversible steps of GTP hydrolysis and peptide bond formation. To study such reactions, transient kinetic techniques are particularly suitable, as they can reveal sequences of interactions and allow monitoring the formation and consumption of the reaction intermediates of protein synthesis in real time.Rapid kinetic analysis has been utilized to study essentially every step of proteins synthesis. In the fi rst part of this review, we consider the basics of transient kinetic techniques. The second part gives an overview of two cases where the results of rapid kinetic studies provided essential insights into the mechanisms of partial reactions of protein synthesis. We will describe how rapid kinetic analyses contributed to understanding the mechanisms of chemical reactions catalyzed by the active site on the 50S ribosomal subunit, i.e., peptide bond formation and peptidyl-tRNA hydrolysis, during the elongation and termination phases of protein synthesis,

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Transition State Chirality and Role of the Vicinal Hydroxyl in the Ribosomal Peptidyl Transferase Reaction

Cited by 15 publications

References 43 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

A structural view on the mechanism of the ribosome-catalyzed peptide bond formation

Rapid Kinetic Analysis of Protein Synthesis

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