Glutamine synthetase brings nitrogen into metabolism by condensing ammonia and glutamate, with the aid of ATP, to yield glutamine, ADP, and inorganic phosphate. Here we present five crystal structures of GS complexed with each of two substrates, Glu and AMPPNP (an ATP analog), with a transition-state analogue, L-methionine-S-sulfoximine, and with each of two products, Gln and ADP. GS of the present study is from Salmonella typhimurium, has Mn2+ bound, and is fully unadenylylated. Protein-metal-substrate interactions and small but significant conformational changes induced by substrate binding are defined by Fourier maps. On the basis of these maps, we propose a tentative structure-based enzymatic mechanism of glutamine synthesis with these steps: (1) ATP binds first at the top of the funnel-shaped active site cavity, adjacent to the n2 Mn2+; Arg 359 moves toward the Glu binding site. (2) Glu binds adjacent to then, Mn2+ at the bottom of the active site near a flexible loop (residues 324-328). As proposed earlier by Meister and others, Glu attacks the gamma-phosphorus atom of ATP to produce gamma-glutamyl phosphate and ADP. (3) The presence of ADP (but not ATP) moves Arg 339 toward the P(i) site, perhaps stabilizing the gamma-glutamyl phosphate, and moves Asp 50' of the adjacent subunit toward a putative ammonium ion site, enhancing binding of this third substrate. Deprotonation of the ammonium ion, perhaps by Asp 50', permits the resulting active species, ammonia, to attack the gamma-glutamyl phosphate, forming a tetrahedral intermediate. (4) This tetrahedral intermediate stabilizes the Glu binding loop, residues 324-328, because of the interaction of the positive charge on the gamma-amino group of the intermediate with the negative charge on the side chain of Glu 327, closing the path of Glu entry through the bottom of the active site funnel. (4) Phosphate leaves through the top of the active site and a proton from the gamma-amino group of the tetrahedral intermediate is lost, perhaps to Glu 327, to yield Gln. The absence of electrostatic interaction between Glu 327 and the product Gln permits the segment (residues 324-328) to open, allowing Gln to leave through the bottom of the active site and Glu to enter for the next catalytic cycle. These five crystal structures of GS-substrate complexes also suggest possible mechanisms for other reactions catalyzed by GS.
