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Calculations of Reaction Mechanisms and Entropic Effects in Enzyme Catalysis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.ORCID iD: 0000-0002-0750-8865
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
Keywords [en]
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: urn:nbn:se:uu:diva-316497ISBN: 978-91-554-9831-3 (print)OAI: oai:DiVA.org:uu-316497DiVA, id: diva2:1077962
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
List of papers
1. Chemical reaction mechanisms in solution from brute force computational Arrhenius plots
Open this publication in new window or tab >>Chemical reaction mechanisms in solution from brute force computational Arrhenius plots
2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7293Article in journal (Refereed) Published
Abstract [en]

Decomposition of activation free energies of chemical reactions, into enthalpic and entropic components, can provide invaluable signatures of mechanistic pathways both in solution and in enzymes. Owing to the large number of degrees of freedom involved in such wcondensed-phase reactions, the extensive configurational sampling needed for reliable entropy estimates is still beyond the scope of quantum chemical calculations. Here we show, for the hydrolytic deamination of cytidine and dihydrocytidine in water, how direct computer simulations of the temperature dependence of free energy profiles can be used to extract very accurate thermodynamic activation parameters. The simulations are based on empirical valence bond models, and we demonstrate that the energetics obtained is insensitive to whether these are calibrated by quantum mechanical calculations or experimental data. The thermodynamic activation parameters are in remarkable agreement with experiment results and allow discrimination among alternative mechanisms, as well as rationalization of their different activation enthalpies and entropies.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-259120 (URN)10.1038/ncomms8293 (DOI)000357171100007 ()26028237 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2015-07-28 Created: 2015-07-27 Last updated: 2017-12-04Bibliographically approved
2. Enzyme catalysis by entropy without Circe effect
Open this publication in new window or tab >>Enzyme catalysis by entropy without Circe effect
2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 9, p. 2406-2411Article in journal (Refereed) Published
Abstract [en]

Entropic effects have often been invoked to explain the extraordinary catalytic power of enzymes. In particular, the hypothesis that enzymes can use part of the substrate-binding free energy to reduce the entropic penalty associated with the subsequent chemical transformation has been very influential. The enzymatic reaction of cytidine deaminase appears to be a distinct example. Here, substrate binding is associated with a significant entropy loss that closely matches the activation entropy penalty for the uncatalyzed reaction inwater, whereas the activation entropy for the rate-limiting catalytic step in the enzyme is close to zero. Herein, we report extensive computer simulations of the cytidine deaminase reaction and its temperature dependence. The energetics of the catalytic reaction is first evaluated by density functional theory calculations. These results are then used to parametrize an empirical valence bond description of the reaction, which allows efficient sampling by molecular dynamics simulations and computation of Arrhenius plots. The thermodynamic activation parameters calculated by this approach are in excellent agreement with experimental data and indeed show an activation entropy close to zero for the rate-limiting transition state. However, the origin of this effect is a change of reaction mechanism compared the uncatalyzed reaction. The enzyme operates by hydroxide ion attack, which is intrinsically associated with a favorable activation entropy. Hence, this has little to do with utilization of binding free energy to pay the entropic penalty but rather reflects how a preorganized active site can stabilize a reaction path that is not operational in solution.

Keywords
cytidine deaminase, density functional theory, empirical valence bond method, computational Arrhenius plots
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-282308 (URN)10.1073/pnas.1521020113 (DOI)000371204500044 ()26755610 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationeSSENCE - An eScience CollaborationSwedish National Infrastructure for Computing (SNIC)
Available from: 2016-04-05 Created: 2016-04-05 Last updated: 2018-01-10Bibliographically approved
3. Peptide Release on the Ribosome Involves Substrate-Assisted Base Catalysis
Open this publication in new window or tab >>Peptide Release on the Ribosome Involves Substrate-Assisted Base Catalysis
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.

Keywords
ribosome, translation termination, release factor, peptidyl-tRNA hydrolysis, density functional theory
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-312638 (URN)10.1021/acscatal.6b02842 (DOI)000389399400051 ()
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
4. Quantum Mechanical Calculations on Alternative Mechanisms for Peptide Bond Formation on the Ribosome
Open this publication in new window or tab >>Quantum Mechanical Calculations on Alternative Mechanisms for Peptide Bond Formation on the Ribosome
(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.

Keywords
Ribosome, peptide bond formation, peptidyl-tRNA hydrolysis, density functional theory
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-316496 (URN)
Funder
Swedish Research Council
Available from: 2017-03-01 Created: 2017-03-01 Last updated: 2017-03-13

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