Enzyme Architecture : Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
Kulkarni, Yashraj S ; Liao, Qinghua ; Petrović, Dušan ; Krüger, Dennis M ; Strodel, Birgit ; Amyes, Tina L ; Richard, John P and Kamerlin, Shina C L LU
(2017)
In Journal of the American Chemical Society
139(30).
p.10514-10525
- Abstract
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 (I170 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... (More)
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 (I170 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 I170A, L230A, and I170A/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.
(Less)
- author
- Kulkarni, Yashraj S
; Liao, Qinghua
; Petrović, Dušan
; Krüger, Dennis M
; Strodel, Birgit
; Amyes, Tina L
; Richard, John P
and Kamerlin, Shina C L
LU
- publishing date
- 2017-08-02
- type
- Contribution to journal
- publication status
- published
- keywords
- Biocatalysis, Dihydroxyacetone Phosphate/chemistry, Glyceraldehyde 3-Phosphate/chemistry, Hydrophobic and Hydrophilic Interactions, Molecular Conformation, Molecular Dynamics Simulation, Saccharomyces cerevisiae/enzymology, Thermodynamics, Triose-Phosphate Isomerase/chemistry
- in
- Journal of the American Chemical Society
- volume
- 139
- issue
- 30
- pages
- 12 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:28683550
- scopus:85026808614
- ISSN
- 1520-5126
- DOI
- 10.1021/jacs.7b05576
- language
- English
- LU publication?
- no
- id
- 369b625d-9325-43fc-b87f-62d4de6bc218
- date added to LUP
- 2025-01-11 21:24:26
- date last changed
- 2025-05-04 12:14:18
@article{369b625d-9325-43fc-b87f-62d4de6bc218,
abstract = {{<p>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 (I170 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 I170A, L230A, and I170A/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.</p>}},
author = {{Kulkarni, Yashraj S and Liao, Qinghua and Petrović, Dušan and Krüger, Dennis M and Strodel, Birgit and Amyes, Tina L and Richard, John P and Kamerlin, Shina C L}},
issn = {{1520-5126}},
keywords = {{Biocatalysis; Dihydroxyacetone Phosphate/chemistry; Glyceraldehyde 3-Phosphate/chemistry; Hydrophobic and Hydrophilic Interactions; Molecular Conformation; Molecular Dynamics Simulation; Saccharomyces cerevisiae/enzymology; Thermodynamics; Triose-Phosphate Isomerase/chemistry}},
language = {{eng}},
month = {{08}},
number = {{30}},
pages = {{10514--10525}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Journal of the American Chemical Society}},
title = {{Enzyme Architecture : Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase}},
url = {{http://dx.doi.org/10.1021/jacs.7b05576}},
doi = {{10.1021/jacs.7b05576}},
volume = {{139}},
year = {{2017}},
}