Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

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Mhasal, Anil RhanuUppsala universitet,Biokemi (author)

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

Article/chapterEnglish2020

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2020-09-03
AMER CHEMICAL SOC,2020
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LIBRIS-ID:oai:DiVA.org:uu-423917
https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-423917URI
https://doi.org/10.1021/acscatal.0c02757DOI

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Language:English
Summary in:English

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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 center dot 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.

Subject headings and genre

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
NATURVETENSKAP Kemi Organisk kemi hsv//swe
NATURAL SCIENCES Chemical Sciences Organic Chemistry hsv//eng
glycerol-3-phosphate dehydrogenase
loop dynamics
transition state stabilization
empirical valence bond
Hamiltonian replica exchange

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Romero-Rivera, AdrianUppsala universitet,Biokemi(Swepub:uu)adrro578 (author)
Mydy, Lisa S.SUNY Buffalo, Jacobs Sch Med & Biomed Sci, Dept Biol Struct, Buffalo, NY 14203 USA. (author)
Cristobal, Judith R.SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA. (author)
Gulick, Andrew M.SUNY Buffalo, Jacobs Sch Med & Biomed Sci, Dept Biol Struct, Buffalo, NY 14203 USA. (author)
Richard, John P.SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA. (author)
Kamerlin, Shina C. Lynn,1981-Uppsala universitet,Institutionen för kemi - BMC(Swepub:uu)lynka392 (author)
Uppsala universitetBiokemi (creator_code:org_t)

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In:ACS Catalysis: AMER CHEMICAL SOC10:19, s. 11253-112672155-5435

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