Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus. Direct evidence for a flip-flop mechanism.
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.
Moniot
Sébastien
S
Laboratoire de Cristallographie et de Modélisation des Matériaux Minéraux et Biologiques, UMR CNRS-Université Henri Poincaré (UHP) 7036.
Bruno
Stefano
S
Vonrhein
Clemens
C
Didierjean
Claude
C
Boschi-Muller
Sandrine
S
Vas
Mária
M
Bricogne
Gérard
G
Branlant
Guy
G
Mozzarelli
Andrea
A
Corbier
Catherine
C
D018360:Crystallography, X-Ray / Q000379:methods; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism*; D008968:Molecular Conformation; D009154:Mutation; D009243:NAD / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D010710:Phosphates; D009243:NAD; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)