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Enzyme catalysis by entropy without Circe effect
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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.
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, 2406-2411 p.Article in journal (Refereed) Published
Resource type
Text
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.

Place, publisher, year, edition, pages
2016. Vol. 113, no 9, 2406-2411 p.
Keyword [en]
cytidine deaminase, density functional theory, empirical valence bond method, computational Arrhenius plots
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-282308DOI: 10.1073/pnas.1521020113ISI: 000371204500044PubMedID: 26755610OAI: oai:DiVA.org:uu-282308DiVA: diva2:916865
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: 2017-11-30Bibliographically 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. 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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Kazemi, MasoudÅqvist, Johan

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