Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus: Direct evidence for a flip-flop mechanism

Sébastien Moniot, Stefano Bruno, Clemens Vonrhein, Claude Didierjean, Sandrine Boschi-Muller, Mária Vas, Gérard Bricogne, Guy Branlant, Andrea Mozzarelli, Catherine Corbier

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Abstract

The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8 Å 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.

Original languageEnglish
Pages (from-to)21693-21702
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number31
DOIs
Publication statusPublished - Aug 1 2008

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Moniot, S., Bruno, S., Vonrhein, C., Didierjean, C., Boschi-Muller, S., Vas, M., Bricogne, G., Branlant, G., Mozzarelli, A., & Corbier, C. (2008). Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus: Direct evidence for a flip-flop mechanism. Journal of Biological Chemistry, 283(31), 21693-21702. https://doi.org/10.1074/jbc.M802286200