User:Wayne Chang

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Assignment 12: IVC: Ammonium Binding Site

Mapping the Ammonium binding site and explaining how it contributes to catalysis.

Chang, Yu-Wei and Kaushal, Pankaj.

BIOL 430: Biological Chemistry.

University of Maryland, Baltimore County (UMBC).

Glutamine Synthetase (GS) catalyzes the ATP dependent condensation of glutamate and ammonia. The reaction produces glutamine, ADP, and an inorganic phosphate group^[1]:

Glutamate + ATP + NH₄⁺ → Glutamine + ADP + Pi

Active Site Structure

Bacterial GS consists of with 622 symmetry. There are 12 funnel shaped active sites located at the interface of each subunit. The is open at the top as well as at the bottom and is about 30 Å wide at its top, about 10 Å wide at the bottom. Each funnel consists of 4 binding sites for the ligands ATP, Mn, Glutamate, and Ammonium ion all of which participate in the reaction GS catalyzes(color coding of binding sites in the funnel: Glutamate, ATP, Mn²⁺, NH₄⁺). ATP binds at the top of the active site, while glutamate binds at the bottom. GS is a trimetallic enzyme, binding two divalent ions (Mn²⁺ or Mg²⁺) and one monovalent ion (NH₄⁺).^[1]

Ammonium binding site - a Negatively charged pocket

The four residues that create the ammonium binding site are . Residues Serine 53 and Asp 50 are found on the adjacent chain which explains the apostrophe after their numbers. All four are negatively charged which is important for the ammonium ion to bind favorably with GS and attack y-glutamyl phosphate. If a water molecule instead of an ammonium ion were to attack y-glutamyl phosphate, the product would be glutamate instead of glutamine. In addition, a negatively charged pocket serves to provide stability with the positive ammonium ion in the active conformation. The proximity of the residues to the glutamate binding site as well as the affinity for the positive ammonium molecule to the negative residues helps to catalyze the reaction.^[1]

Catalysis

The reaction is catalyzed in a mulit-step process. Available evidence shows that ammonium ion, and not ammonia, is the initial substrate in the reaction. A deprotonated ammonia completes the reaction after an ammonium ion binds to the negatively charged pocket of GS. In the first step, glutamate reacts with ATP to produce ADP and the intermediate y-glutamyl phosphate. ADP binds to Asp 50’. The ADP binding induces Asp 50’ to move toward the ammonium binding site to enhance ammonium binding. Ammonium is then deprotonated, perhaps by Asp 50’, to form ammonia that reacts with the intermediate y-glutamyl phosphate, yielding glutamine. ^[1][2]

References

1. ↑ 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[1].
2. ↑ Liaw SH, Eisenberg D. Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes. Biochemistry. 1994 Jan 25;33(3):675-81.