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Quantum Mechanical Calculations on Alternative Mechanisms for Peptide Bond Formation on the Ribosome
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.ORCID iD: 0000-0002-0750-8865
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.ORCID iD: 0000-0003-2091-0610
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Peptide bond formation on the ribosome involves nucleophilic attack of the terminal amine of the newly delivered aminoacyl-tRNA on the ester bond of the peptidyl-tRNA carrying the growing peptide. The reaction takes place in the peptidyl transferase center (PTC) on the large ribosomal subunit during the elongation phase of protein synthesis. This peptidyl transfer reaction depends only on the protonation state of the α-amino group and exhibits a large kinetic solvent isotope effect (KSIE ~8). This is clearly different from the experimental signature of peptidyl-tRNA hydrolysis which is also catalyzed by the PTC. For peptidyl-tRNA hydrolysis, the magnitude of the KSIE is ~4 and the pH-rate profile has a slope of one suggesting that this reaction involves base catalysis. However, it is not clear why these reactions should proceed with different mechanisms, as is evident from the experimental data. One explanation is that two competing mechanisms may be operational in the PTC. Herein, we explored this possibility by re-examining the previously proposed proton shuttle mechanism and testing the feasibility of general base catalysis also for peptide bond formation. We employed a large cluster model of the active site and different reaction mechanisms were evaluated by density functional theory (DFT) calculations. In these calculations, the proton shuttle and general base mechanisms both yield activation energies comparable to the experimental values. However, only the proton shuttle mechanism is found to be consistent with the experimentally observed pH-rate profile and the KSIE. This suggests that the PTC promotes the proton shuttle mechanism for peptide bond formation, while prohibiting general base catalysis, although the detailed mechanism by which general base catalysis is prohibited remains unclear.

Keyword [en]
Ribosome, peptide bond formation, peptidyl-tRNA hydrolysis, density functional theory
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-316496OAI: oai:DiVA.org:uu-316496DiVA: diva2:1077960
Funder
Swedish Research Council
Available from: 2017-03-01 Created: 2017-03-01 Last updated: 2017-03-13
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