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
Kulkarni, Yashraj S ; Amyes, Tina L ; Richard, John P and Kamerlin, Shina C L LU
(2019)
In Journal of the American Chemical Society
141(40).
p.16139-16150
- Abstract
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... (More)
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 ΔG° 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.
(Less)
- author
- Kulkarni, Yashraj S
; Amyes, Tina L
; Richard, John P
and Kamerlin, Shina C L
LU
- publishing date
- 2019-10-09
- type
- Contribution to journal
- publication status
- published
- keywords
- Amino Acid Substitution, Amino Acids/chemistry, Catalysis, Catalytic Domain, Dihydroxyacetone Phosphate/chemistry, Glyceraldehyde 3-Phosphate/chemistry, Kinetics, Models, Molecular, Mutation, Protons, Thermodynamics, Triose-Phosphate Isomerase/chemistry
- in
- Journal of the American Chemical Society
- volume
- 141
- issue
- 40
- pages
- 12 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:31508957
- scopus:85072967186
- ISSN
- 1520-5126
- DOI
- 10.1021/jacs.9b08713
- language
- English
- LU publication?
- no
- id
- b1de8ec1-fda0-4eaf-a4ff-debab0f9b418
- date added to LUP
- 2025-01-11 20:26:53
- date last changed
- 2025-05-18 14:51:32
@article{b1de8ec1-fda0-4eaf-a4ff-debab0f9b418,
abstract = {{<p>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 ΔG° 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.</p>}},
author = {{Kulkarni, Yashraj S and Amyes, Tina L and Richard, John P and Kamerlin, Shina C L}},
issn = {{1520-5126}},
keywords = {{Amino Acid Substitution; Amino Acids/chemistry; Catalysis; Catalytic Domain; Dihydroxyacetone Phosphate/chemistry; Glyceraldehyde 3-Phosphate/chemistry; Kinetics; Models, Molecular; Mutation; Protons; Thermodynamics; Triose-Phosphate Isomerase/chemistry}},
language = {{eng}},
month = {{10}},
number = {{40}},
pages = {{16139--16150}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Journal of the American Chemical Society}},
title = {{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}},
url = {{http://dx.doi.org/10.1021/jacs.9b08713}},
doi = {{10.1021/jacs.9b08713}},
volume = {{141}},
year = {{2019}},
}