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From Proteopedia

Revision as of 03:39, 20 December 2008

Background
Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. ^[1] The reaction occurs in two steps with γ-glutamyl phosphate as an intermediate and is used by bacteria to introduce reduced nitrogen into cellular metabolism. GS is a dodecamer formed from two face-to-face hexameric rings of subunits, with 12 active sites formed between monomers. ^[2] Each active site is described as a bifunnel allowing ATP and glutamate to bind at opposite ends. ^[3] Divalent metal ions n1 and n2 (Mg²⁺ or Mn²⁺) bind at the center of the bifunnel and are important for catalysis and stutructural stability.

Overall Mechanism
The first step is the formation of the activated intermediate γ-glutamyl phosphate. The n2 ion coordinates the phosphate oxygens of ATP to allow phosphoryl transfer to the γ-carboxylate group of glutatmate, yeilding the intermediate. ^[3] The second step is the attack on the intermediate by ammonia therefore releasing free phosphate to yield glutamine.

ATP binding site
ATP binds at the top of the active site cavity and the glutamate binds at the bottom, adjacent to the n1 ion ^[1]. The movement of Arg 359 toward the glutamate site, induced by ATP binding, increases the binding affinity of glutamate. The active site of GS is located at the subunit interface (which contains n1 & n2) and is constituted mainly by the C domain of one subunit.(figure below) ^[1]

D. Eisneberg et al / Biochimica et Biophysica Acta 1477 (2000) 124

Involving Residues
Most residues involved in enzymatic catalysis are located at the C domain but Asp50 is contributed from the N domain of the other subunit. The binding of ADP induces Asp50 in order to enhance the ammonium binding, and then to deprotonate the ammonium ion to form the active ammonia to attack the gamma-glutamyl phosphate.

More Catalytic Residues^[1]

Residue	Role in enzymatic mechanism
Arg-321	Coordinates the carboxylate of glutamate
Glu-327	Closes active site and shields intermediate from hydrolysis
His-269	Coordinates the n2 ion
Glu-220	Coordinates the n1 ion
Asp-50	Increases the affinity for ammonium binding

References

1. ↑ ^{1.0 1.1 1.2 1.3} Liaw, S-H, et.al.,Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site Protein Sci. 1995 4: 2358-2365
2. ↑ Gill, H & Eisenberg, D., Biochemistry 2001 40: 1903-1912
3. ↑ ^{3.0 3.1} Eisenberg, D., et.al., Structure-function relationships of glutamine synthetases, Biochim Biophys Acta 2000: 1477, 122-145.