Document Detail

Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus. Direct evidence for a flip-flop mechanism.
MedLine Citation:
PMID:  18480053     Owner:  NLM     Status:  MEDLINE    
The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8A 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 P(i)" 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.
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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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-05-14
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  283     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2008 Aug 
Date Detail:
Created Date:  2008-07-28     Completed Date:  2008-09-22     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  21693-702     Citation Subset:  IM    
Laboratoire de Cristallographie et de Modélisation des Matériaux Minéraux et Biologiques, UMR CNRS-Université Henri Poincaré (UHP) 7036.
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MeSH Terms
Crystallography, X-Ray / methods
Escherichia coli / metabolism
Geobacillus stearothermophilus / enzymology*
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / metabolism*
Molecular Conformation
NAD / chemistry
Phosphates / chemistry
Protein Conformation
Spectrophotometry / methods
Substrate Specificity
X-Ray Diffraction
Reg. No./Substance:
0/Phosphates; 53-84-9/NAD; EC Dehydrogenase (Phosphorylating)

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