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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
In thesis
1. Calculations of Reaction Mechanisms and Entropic Effects in Enzyme Catalysis
Open this publication in new window or tab >>Calculations of Reaction Mechanisms and Entropic Effects in Enzyme Catalysis
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost.

Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell.

The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization.

In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water.

Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1482
Keyword
Enzyme catalysis, Entropy, Cytidine deamination, Ribosome, Peptidyl-tRNA hydrolysis, Peptide bond formation, Empirical valence bond method, Density functional theory
National Category
Biochemistry and Molecular Biology Theoretical Chemistry
Research subject
Biology with specialization in Structural Biology; Biochemistry; Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-316497 (URN)978-91-554-9831-3 (ISBN)
Public defence
2017-04-21, B41, Biomedicinska Centrum (BMC) Husarg. 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-03-27 Created: 2017-03-01 Last updated: 2017-03-30

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