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Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Forschungszentrum Julich, Inst Complex Syst Struct Biochem, D-52425 Julich, Germany..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2017 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 30, p. 10514-10525Article in journal (Refereed) Published
Abstract [en]

Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (1170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild type TIM and its 1170A, L230A, and 1170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017. Vol. 139, no 30, p. 10514-10525
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-334051DOI: 10.1021/jacs.7b05576ISI: 000407089500046PubMedID: 28683550OAI: oai:DiVA.org:uu-334051DiVA, id: diva2:1158836
Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2019-12-04Bibliographically approved
In thesis
1. Computational Modeling of the Structure, Function and Dynamics of Biomolecular Systems
Open this publication in new window or tab >>Computational Modeling of the Structure, Function and Dynamics of Biomolecular Systems
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are a structurally diverse and functionally versatile class of biomolecules. They perform a variety of life-sustaining biological processes with utmost efficiency. A profound understanding of protein function requires knowledge of its structure. Experimentally determined protein structures can serve as a starting point for computer simulations in order to study their dynamic behavior at a molecular level. In this thesis, computational methods have been used to understand structure-function relationships in two classes of proteins - intrinsically disordered proteins (IDP) and enzymes.

Misfolding and subsequent aggregation of the amyloid beta (Aβ) peptide, an IDP, is associated with the progression of Alzheimer’s disease. Besides enriching our understanding of structural dynamics, computational studies on a medically relevant IDP such as Aβ can potentially guide therapeutic development. In the present work, binding interactions of the monomeric form of this peptide with biologically relevant molecular species such as divalent metal ions (Zn2+, Cu2+, Mn2+) and amphiphilic surfactants were characterized using long timescale molecular dynamics (MD) simulations. Among the metal ions, while Zn2+ and Cu2+ maintained coordination to a well-defined binding site in Aβ, Mn2+-binding was observed to be comparatively weak and transient. Surfactants with charged headgroups displayed strong binding interaction with Aβ. Complemented by biophysical experiments, these studies provided a multifaceted perspective of Aβ interactions with the partner molecules.

Triosephosphate isomerase (TIM), a highly evolved and catalytically proficient enzyme, was studied using empirical valence bond (EVB) calculations to obtain deeper insights into the catalytic reaction mechanism. Multiple structural features of TIM such as the flexible loop and preorganized active site residues were investigated for their role in enzyme catalysis. The effect of substrate binding was also studied using truncated substrates. Finally, using enhanced sampling methods, dynamic behavior of the catalytically important loop 6 was characterized. The importance of structural stability and flexibility on protein function was illustrated by the work presented in this thesis, thus furthering our scientific understanding of proteins at a molecular level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1885
Keywords
Molecular Dynamics, Empirical Valence Bond, Enzyme Catalysis, Amyloid Beta, Aβ, Triosephosphate Isomerase, TIM, Computational Biochemistry, Computational Enzymology
National Category
Biochemistry and Molecular Biology Theoretical Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-398169 (URN)978-91-513-0828-9 (ISBN)
Public defence
2020-02-05, B21, BMC, Husargatan 3, Uppsala, 13:15 (English)
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Supervisors
Available from: 2020-01-14 Created: 2019-12-04 Last updated: 2020-01-14

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Kulkarni, Yashraj S.Krüger, Dennis M.Kamerlin, Shina C. L.

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