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

Nature volume 476, pages 351–354 (2011)Cite this article

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Abstract

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-factor-dependent 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 2′-OH group of A76 of the peptidyl - tRNA substrate seems to have a key role in peptide release3. According to computational analysis4 , the 2′-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 formation4,5,6. Here we report kinetic solvent isotope effects and proton inventories (reaction rates measured in buffers with increasing content of deuterated water, D2O) 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 eight-membered proton shuttle in the transition state5. Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase centre.

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Figure 1: RF2-catalysed hydrolysis of fMet-tRNA fMet on the ribosome.
Figure 2: KSIE and pH dependence of the hydrolysis reaction.
Figure 3: KSIE of the peptidyl transfer reaction.

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Acknowledgements

We thank H.-H. Limbach for discussions and advice. This work was supported by the Deutsche Forschungsgemeinschaft (M.V.R. and W.W.).

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  1. Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany

    Stephan Kuhlenkoetter, Wolfgang Wintermeyer & Marina V. Rodnina

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  1. Stephan Kuhlenkoetter
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  2. Wolfgang Wintermeyer
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  3. Marina V. Rodnina
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Contributions

S.K., W.W. and M.V.R. conceived the study and designed experiments. S.K. performed experiments. All three authors discussed results and wrote the paper.

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Correspondence to Marina V. Rodnina.

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The authors declare no competing financial interests.

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Kuhlenkoetter, S., Wintermeyer, W. & Rodnina, M. Different substrate-dependent transition states in the active site of the ribosome. Nature 476, 351–354 (2011). https://doi.org/10.1038/nature10247

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