Dataset Information

Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.

ABSTRACT: The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation (Gua+) [J. Am. Chem. Soc. 2015, 137, 5312-5315]. The R269A mutant shows no detectable activity toward reduction of glycolaldehyde (GA), or activation of this reaction by 30 mM HPO32-. We report the unprecedented self-assembly of R269A hlGPDH, dianions (X2- = FPO32-, HPO32-, or SO42-), Gua+ and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants kcat/KGAKXKGua. The linear logarithmic correlation (slope = 1.0) between values of kcat/KGAKX for dianion activation of wildtype hlGPDH-catalyzed reduction of GA and kcat/KGAKXKGua shows that the electrostatic interaction between exogenous dianions and the side chain of R269 is not significantly perturbed by cutting hlGPDH into R269A and Gua+ pieces. The advantage for connection of hlGPDH (R269A mutant + Gua+) and substrate pieces (GA + HPi) pieces, (?GS‡)HPi+E+Gua = 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (?GS‡)GA+HPi = 3.3 kcal/mol, for wildtype hlGPDH-catalyzed reaction of GA + HPi, and for connection of the enzyme pieces, (?GS‡)E+Gua = 2.4 kcal/mol, for Gua+ activation of the R269A hlGPDH-catalyzed reaction of DHAP.

SUBMITTER: Reyes AC

PROVIDER: S-EPMC5291162 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.

Reyes Archie C AC Amyes Tina L TL Richard John P JP

Journal of the American Chemical Society 20161110 46

The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation ...[more]

PMID: 27792325

Similar Datasets

Project description:Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition-state stabilization. While the human form (hlGPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hlGPDH and its variants, as well as providing a crystal structure of the binary hlGPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted hlGPDH and investigated the effect of mutations on catalysis from a detailed structural study. In particular, the K120A and R269A variants increase both the volume and solvent exposure of the active site, with concomitant loss of catalytic activity. In addition, the R269 side chain interacts with both the Q295 side chain on the catalytic loop, and the substrate phosphodianion. Our structural data and simulations illustrate the critical role of this side chain in facilitating the closure of hlGPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297). This, in turn, rationalizes a tremendous 41,000 fold decrease experimentally in the turnover number, k cat, upon truncating this residue, as loop closure is essential for both correct positioning of key catalytic residues in the active site, as well as sequestering the active site from the solvent. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of allosteric inhibitors targeting this biomedically important enzyme.

Dataset Information

Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.

Publications

Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets