Abstract
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, kcat, 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.
Highlights
Human liver glycerol-3-phosphate dehydrogenaseis a dimeric enzyme that catalyzes the NADH-dependent reduction of cytosolic dihydroxyacetone phosphate (DHAP)to glycerol-3-phosphate (G3P), a compound which serves as a key precursor for lipid synthesis.[1]
There have been a range of structural analyses of glycerol-3-phosphate dehydrogenases from different organisms that provide important information into the overall structure of the enzyme[5,11,62], as well as biochemical and kinetic studies of the catalytic activity of different GPDHs,[1,5,11,63] and the activation of GPDH by phosphite dianions.[9,64]
Carbonyl group, are positioned to act as viable proton donors (Figure 1, based on distances between the nitrogen atom of the lysine side chain, and the carbonyl oxygen of DHAP in PDB ID: 6E905,6). It was recently suggested[5] that the side chain alkyl ammonium cation of K120, which is ion paired with the side chain of D260, can function as a Brønsted acid and donate a proton to the carbonyl oxygen of the substrate. This was based on the fact that in the structure of the nonproductive ternary complex of hlGPDH·NAD·DHAP (PDB ID: 6E905,6), the Nε atom of K120 lies nearly coplanar with the plane defined by the trigonal C O bond of DHAP, facilitating proton transfer from K120 to the carbonyl oxygen, whereas the corresponding Nε atom of K204 lies well below this plane and was judged to be less likely to participate in the protonation of carbonyl oxygen (Figure 1)
Summary
Human liver glycerol-3-phosphate dehydrogenase (hlGPDH)is a dimeric enzyme that catalyzes the NADH-dependent reduction of cytosolic dihydroxyacetone phosphate (DHAP)to glycerol-3-phosphate (G3P), a compound which serves as a key precursor for lipid synthesis.[1]. Truncation of Q295 and R269 to alanine (Q295A and R269A) has been shown to lead to 3.0 and 9.1 kcal·mol−1 destabilization of the TS for the reduction of DHAP,[11] respectively (in terms of decrease in kcat/KM), and the R269A variant in particular has been shown to lead to a 41,000 fold decrease in the turnover number (kcat).[5,20] This impaired catalytic activity has been ascribed to the electrostatic destabilization of the TS in the substituted enzymes.[11] while electrostatic destabilization through the loss of the ion pair between R269 and the substrate phosphodianion will clearly play a role in facilitating the loss of activity upon truncation of this residue, additional effects from structural perturbations and incomplete closure of the flexible loop upon the truncation of the arginine side chain cannot be ruled out This possibility is supported by the fact that single point mutations in enzymes have been shown to bring about large conformational changes that can alter their functionally relevant motions in a catalytically deleterious way.[21−23] We note that for related enzymes, such as dihydrofolate reductase[24] and TIM,[25] such loop motions have been shown to be partially rate-limiting for the chemical step
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