Crystal Structure of Two Ternary Complexes of Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from Bacillus stearothermophilus with NAD and d-Glyceraldehyde 3-Phosphate

Abstract

The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate, D-glyceraldehyde 3-phosphate (D-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys 149 , was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals of the corresponding binary complexes (enzyme⅐NAD) in a solution containing G3P. The structures of the two binary and the two ternary complexes are presented. The D-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form, its C-3 phosphate group being positioned in the P s site and not in the P i site. Its C-1 carbonyl oxygen points toward the essential His 176 , which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that the structures reported here are representative of a productive enzyme⅐NAD⅐D-G3P complex in the ground state (Michaelis complex). Phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH)1 are tetrameric enzymes that catalyze reversibly the oxidative phosphorylation of D-glyceraldehyde 3-phosphate (D-G3P) into 1,3-diphosphoglycerate (1,3-dPG) in the presence of cofactor NAD(P) via a two-step chemical mechanism. This mechanism has been well documented for the homotetrameric GAPDHs involved in glycolysis (1). First, the acylation step leads to the formation of a thioacylenzyme intermediate and NADH. This step includes: 1) the binding of D-G3P to the binary complex GAPDH⅐NAD; 2) the formation of a covalent thiohemiacetal intermediate with D-G3P; and 3) the hydride transfer from the thiohemiacetal intermediate toward the C-4 position of the nicotinamide ring of NAD. Second, the phosphorylation step consists of a nucleophilic attack of inorganic phosphate toward the thioacylenzyme intermediate that leads to formation of 1,3-dPG. This step, which is rate-limiting, includes the binding of inorganic phosphate to the enzyme intermediate, possibly preceded by an isomerization step

The Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Is Critical for Viable Pollen Development in Arabidopsis

“…This Cys residue has also been reported to be important in signaling mechanisms such as S-nitrosylationdependent apoptotic cell death cascades (Hara et al, 2005). To ensure that only the glycolytic functions of GAPCp were altered, other important amino acids for GAPCp1 glycolytic activity were also substituted (H263A and R318E; Didierjean et al, 2003). None of these mutant constructs was able to complement the male sterile phenotype of gapcp1gapcp2, thus confirming that the glycolytic activity of GAPCp is essential for correct pollen development.…”

“…Because of this, we transformed gapcp1gapcp2 with GAPCps inactivated in their catalytic domain. Cys-236 has been described as being essential for catalytic activity (Didierjean et al, 2003). This Cys residue has also been reported to be important in signaling mechanisms such as S-nitrosylationdependent apoptotic cell death cascades (Hara et al, 2005).…”

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

“…4A and Table 2). The first conformation, called conformation A, is strictly equivalent to the conformation described in the holo-structure (10,16). In this conformation, the loop participates in the binding of a sul- fate anion in the "classic" P i site.…”

“…C, stereoscopic view of the superimposition between this model (green) and the Michaelis ternary complex from B. stearothermophilus holo-GAPDH (C149S mutant, in blue) (1NQO) (16).…”

“…Recently, using mutants of the active site cysteine, Didierjean et al (16) obtained the structures of ternary complexes from B. stearothermophilus GAPDH with NAD ϩ and the physiological substrate D-G3P, the phosphate group of which was found to be bound at the P s site. This result strongly suggests that the P s site constitutes the binding site for the C3P group of the substrate but does not, however, exclude the possibility that the substrate could display different interactions especially with regard to its C3P group, once the covalent bond is formed between Cys-149 and D-G3P.…”