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PDBsum entry 1nq5
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Oxidoreductase
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PDB id
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1nq5
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-Phosphate dehydrogenase from bacillus stearothermophilus with NAD and d-Glyceraldehyde 3-Phosphate.
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Authors
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C.Didierjean,
C.Corbier,
M.Fatih,
F.Favier,
S.Boschi-Muller,
G.Branlant,
A.Aubry.
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Ref.
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J Biol Chem, 2003,
278,
12968-12976.
[DOI no: ]
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PubMed id
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Abstract
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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 (enzyme.NAD) 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 enzyme.NAD.D-G3P complex in the ground state (Michaelis complex).
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Figure 1.
Fig. 1. Schematic representation of the postulated
catalytic mechanism of phosphorylating GAPDHs. The catalytic
mechanism can be divided into two steps. In the acylation step,
Cys149 and His176 form an ion pair in holo-GAPDH (a). This
decreases the pK[app] of Cys149, thus facilitating the thiolate
attack toward the C-1 of D-G3P. The role of His 176 is also to
stabilize the binding of the substrate in the Michaelis complex
GAPDH·NAD·D-G3P (b), the thiohemiacetal
intermediate (c), and the thioacylenzyme intermediate (d).
His176 also plays a role as a base catalyst facilitating the
hydride transfer from the thiohemiacetal toward the
nicotinamidium of NAD (c). In the phosphorylating step, the
binding of inorganic phosphate to the thioacylenzyme is followed
by its nucleophilic attack toward the thioacyl intermediate (d),
which leads via a sp3-phosphorylated intermediate (e) to the
formation and release of 1,3-dPG (f). His176 is postulated to
stabilize the tetrahedral intermediate (e) and to facilitate, as
an acid (d) or base (e) catalyst, the 1,3-dPG formation. The
exchange cofactor step, which consists of NADH release prior to
NAD and inorganic phosphate binding, remains controversial
(32-40). R' represents the
adenine-ribose-phosphate-phosphate-ribose part of the cofactor,
NAD. R represents the CH(OH)COPO[3] part of the substrate, D-G3P.
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Figure 5.
Fig. 5. Stereoview of the active site in monomer O of the
ternary complexes. A, ternary complex I (C149A mutant GAPDH); B,
ternary complex II (C149A mutant GAPDH). The residues that
interact with the D-G3P molecule are labeled.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2003,
278,
12968-12976)
copyright 2003.
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