Thermal Destabilization of Non-phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from Streptococcus mutans upon Phosphate Binding in the Active Site

Autor: Guy Branlant, Victor N. Orlov, Stéphane Marchal, Denis Arutyunov, Vladimir I. Muronetz, Sophie Rahuel-Clermont
Přispěvatelé: Maturation des ARN et enzymologie moléculaire (MAEM), Cancéropôle du Grand Est-Université Henri Poincaré - Nancy 1 (UHP)-IFR111-Centre National de la Recherche Scientifique (CNRS), Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University (MSU), Mécanismes moléculaires dans les démences neurodégénératives (MMDN), Université de Montpellier (UM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), RAHUEL-CLERMONT, Sophie, Université Montpellier 2 - Sciences et Techniques (UM2)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Rok vydání: 2005
Předmět:
Protein Denaturation
Hot Temperature
Time Factors
Protein Conformation
Dithionitrobenzoic Acid
Dehydrogenase
Substrate analog
Ligands
Biochemistry
Streptococcus mutans
chemistry.chemical_compound
Enzyme Stability
Phosphorylation
[SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry
Molecular Biology/Biochemistry [q-bio.BM]
Ternary complex
Glyceraldehyde 3-phosphate dehydrogenase
0303 health sciences
Calorimetry
Differential Scanning
biology
Sulfates
Chemistry
Circular Dichroism
Hydrolysis
030302 biochemistry & molecular biology
Temperature
Chromatography
Gel
Electrophoresis
Polyacrylamide Gel
Protein Binding
Stereochemistry
Kinetics
Inorganic chemistry
Catalysis
Phosphates
03 medical and health sciences
Differential scanning calorimetry
Chlorides
Cysteine
[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry
Molecular Biology/Biochemistry [q-bio.BM]
Molecular Biology
030304 developmental biology
Binding Sites
Active site
Cell Biology
Phosphate
Mutation
Mutagenesis
Site-Directed
biology.protein
Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
Ultracentrifugation
Zdroj: Journal of Biological Chemistry
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2005, 280 (19), pp.18590-18597. ⟨10.1074/jbc.M414110200⟩
Journal of Biological Chemistry, 2005, 280 (19), pp.18590-18597. ⟨10.1074/jbc.M414110200⟩
ISSN: 0021-9258
1083-351X
Popis: International audience; Catalysis by the NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, a member of the aldehyde dehydrogenase (ALDH) family, relies on a local conformational reorganization of the active site. This rearrangement is promoted by the binding of NADP and is strongly kinetically favored by the formation of the ternary complex enzyme.NADP.substrate. Adiabatic differential scanning calorimetry was used to investigate the effect of ligands on the irreversible thermal denaturation of GAPN. We showed that phosphate binds to GAPN, resulting in the formation of a GAPN.phosphate binary complex characterized by a strongly decreased thermal stability, with a difference of at least 15 degrees C between the maximum temperatures of the thermal transition peaks. The kinetics of phosphate association and dissociation are slow, allowing both free and GAPN.phosphate complexes to be observed by differential scanning calorimetry and to be separated by native polyacrylamide electrophoresis run in phosphate buffer. Analysis of a set of mutants of GAPN strongly suggests that phosphate is bound to the substrate C-3 subsite. In addition, the substrate analog glycerol-3-phosphate has similar effects as does phosphate on the thermal behavior of GAPN. Based on the current knowledge on the catalytic mechanism of GAPN and other ALDHs, we propose that ligand-induced thermal destabilization is a mechanism that provides to ALDHs the required flexibility for an efficient catalysis.
Databáze: OpenAIRE
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