uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Uncovering the Role of Key Active-Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.ORCID iD: 0000-0002-7643-9867
SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA.
SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.ORCID iD: 0000-0002-3190-1173
2019 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 141, no 40, p. 16139-16150Article in journal (Refereed) Published
Abstract [en]

We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol–1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol–1, with a strong linear correlation between ΔG and Δ for all 11 variants, with slopes β = 0.73 (R2 = 0.994) and β = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.

Place, publisher, year, edition, pages
2019. Vol. 141, no 40, p. 16139-16150
National Category
Theoretical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-397177DOI: 10.1021/jacs.9b08713ISI: 000490358900049PubMedID: 31508957OAI: oai:DiVA.org:uu-397177DiVA, id: diva2:1370699
Funder
Swedish Research Council, 2015-04298NIH (National Institute of Health), GM03597NIH (National Institute of Health), GM116921Knut and Alice Wallenberg Foundation, 2013.0124Knut and Alice Wallenberg Foundation, 2018.0140Swedish National Infrastructure for Computing (SNIC), 2017/12-11Swedish National Infrastructure for Computing (SNIC), 2018/2-3Available from: 2019-11-16 Created: 2019-11-16 Last updated: 2019-12-06Bibliographically 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)
Opponent
Supervisors
Available from: 2020-01-14 Created: 2019-12-04 Last updated: 2020-01-14

Open Access in DiVA

The full text will be freely available from 2020-09-11 23:29
Available from 2020-09-11 23:29

Other links

Publisher's full textPubMed

Authority records BETA

Kulkarni, YashrajKamerlin, Shina C. Lynn

Search in DiVA

By author/editor
Kulkarni, YashrajKamerlin, Shina C. Lynn
By organisation
Science for Life Laboratory, SciLifeLabStructural BiologyBiochemistry
In the same journal
Journal of the American Chemical Society
Theoretical Chemistry

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 29 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf