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Trapping of the Thioacylglyceraldehyde-3-phosphate Dehydrogenase Intermediate from Bacillus stearothermophilus

Abstract : The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8Å resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex ( Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968-12976 ) the 206-210 loop is shifted and now forms part of the so-called “new Pi” site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.
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J. Biol. Chem.-2008-Moniot-216...
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Sébastien Moniot, Stefano Bruno, Clemens Vonrhein, Claude Didierjean, Sandrine Boschi-Muller, et al.. Trapping of the Thioacylglyceraldehyde-3-phosphate Dehydrogenase Intermediate from Bacillus stearothermophilus. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2008, 283 (31), pp.21693-21702. ⟨10.1074/jbc.M802286200⟩. ⟨hal-01690593⟩



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