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Peptide Release on the Ribosome Involves Substrate-Assisted Base Catalysis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
Stockholm Univ, Arrhenius Lab, Dept Organ Chem, SE-10691 Stockholm, Sweden..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
2016 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 6, no 12, p. 8432-8439Article in journal (Refereed) Published
Abstract [en]

Termination of protein synthesis on the ribosome involves hydrolysis of the ester bond between the P-site tRNA and the nascent peptide chain. This reaction occurs in the peptidyl transferase center and is triggered by the class I release factors RF1 and RF2 in prokaryotes. Peptidyl-tRNA hydrolysis is pH-dependent, and experimental results suggest that an ionizable group with pK(a) > 9 is involved in the reaction. The nature of this group is, however, unknown. To resolve this problem, we conducted density functional theory calculations using a large cluster model of the peptidyl transferase center. Our calculations reveal that peptidyl-tRNA hydrolysis occurs via a base-catalyzed mechanism with a predicted activation energy of 15.8 kcal mol(-1), which is in good agreement with experimental data. In this mechanism, the P-site A76 2'-OH group is deprotonated and acts as the general base by activating the nucleophilic water molecule. The energy cost of deprotonating the 2'-hydroxyl group at pH 7.5 is estimated to be about 8 kcal mo1(-1), on the basis of its experimental plc in aqueous solution, and this step is predicted to be the source of the observed pH dependence. The proposed mechanism is consistent not only with experimentally derived activation energies but also with the observed kinetic solvent isotope effect.

Place, publisher, year, edition, pages
2016. Vol. 6, no 12, p. 8432-8439
Keyword [en]
ribosome, translation termination, release factor, peptidyl-tRNA hydrolysis, density functional theory
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-312638DOI: 10.1021/acscatal.6b02842ISI: 000389399400051OAI: oai:DiVA.org:uu-312638DiVA, id: diva2:1068828
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationeSSENCE - An eScience CollaborationSwedish National Infrastructure for Computing (SNIC)
Available from: 2017-01-26 Created: 2017-01-12 Last updated: 2017-11-29Bibliographically approved
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. p. 52
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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