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

Abstract : 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.
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https://hal.univ-lorraine.fr/hal-01681520
Contributor : Sophie Rahuel-Clermont <>
Submitted on : Thursday, January 11, 2018 - 4:58:38 PM
Last modification on : Friday, February 26, 2021 - 3:02:02 PM
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Sophie Rahuel-Clermont, Denis Arutyunov, Stephane Marchal, Victor Orlov, Vladimir Muronetz, et al.. Thermal Destabilization of Non-phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from Streptococcus mutans upon Phosphate Binding in the Active Site. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2005, 280 (19), pp.18590-18597. ⟨10.1074/jbc.M414110200⟩. ⟨hal-01681520⟩

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