Dataset Information

Hydride Transfer Catalyzed by Glycerol Phosphate Dehydrogenase: Recruitment of an Acidic Amino Acid Side Chain to Rescue a Damaged Enzyme.

ABSTRACT: K120 of glycerol 3-phosphate dehydrogenase (GPDH) lies close to the carbonyl group of the bound dihydroxyacetone phosphate (DHAP) dianion. pH rate (pH 4.6-9.0) profiles are reported for k_cat and (k_cat/K_m)_dianion for wild type and K120A GPDH-catalyzed reduction of DHAP by NADH, and for (k_cat/K_dK_am) for activation of the variant-catalyzed reduction by CH₃CH₂NH₃⁺, where K_am and K_d are apparent dissociation constants for CH₃CH₂NH₃⁺ and DHAP, respectively. These profiles provide evidence that the K120 side chain cation, which is stabilized by an ion-pairing interaction with the D260 side chain, remains protonated between pH 4.6 and 9.0. The profiles for wild type and K120A variant GPDH show downward breaks at a similar pH value (7.6) that are attributed to protonation of the K204 side chain, which also lies close to the substrate carbonyl oxygen. The pH profiles for (k_cat/K_m)_dianion and (k_cat/K_dK_am) for the K120A variant show that the monoprotonated form of the variant is activated for catalysis by CH₃CH₂NH₃⁺ but has no detectable activity, compared to the diprotonated variant, for unactivated reduction of DHAP. The pH profile for k_cat shows that the monoprotonated K120A variant is active toward reduction of enzyme-bound DHAP, because of activation by a ligand-driven conformational change. Upward breaks in the pH profiles for k_cat and (k_cat/K_m)_dianion for K120A GPDH are attributed to protonation of D260. These breaks are consistent with the functional replacement of K120 by D260, and a plasticity in the catalytic roles of the active site side chains.

SUBMITTER: He R

PROVIDER: S-EPMC7784668 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

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

Hydride Transfer Catalyzed by Glycerol Phosphate Dehydrogenase: Recruitment of an Acidic Amino Acid Side Chain to Rescue a Damaged Enzyme.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets