Glutamine synthetase

Glutamine synthetase (GS) (

Glutamate + ATP + NH₃ → Glutamine + ADP + phosphate

Glutamine synthetase uses ammonia produced by nitrate reduction,

Other reactions may take place via GS. Competition between ammonium ion and water, their binding affinities, and the concentration of ammonium ion, influences glutamine synthesis and glutamine hydrolysis. Glutamine is formed if an ammonium ion attacks the acyl-phosphate intermediate, while glutamate is remade if water attacks the intermediate.^[6][7] Ammonium ion binds more strongly than water to GS due to electrostatic forces between a cation and a negatively charged pocket.^[4] Another possible reaction is upon NH₂OH binding to GS, rather than NH₄+, yields γ-glutamylhydroxamate.^[6][7]

Structure

Glutamine synthetase can be composed of 8, 10, or 12 identical subunits separated into two face-to-face rings.

GS catalyzes the ATP-dependent condensation of glutamate with ammonia to yield glutamine.[4] The hydrolysis of ATP drives^[8] the first step of a two-part, concerted mechanism.^[4][6] ATP phosphorylates glutamate to form ADP and an acyl-phosphate intermediate, γ-glutamyl phosphate, which reacts with ammonia, forming glutamine and inorganic phosphate. ADP and P_i do not dissociate until ammonia binds and glutamine is released.^[6]

ATP binds first to the top of the active site near a cation binding site, while glutamate binds near the second cation binding site at the bottom of the active site.^[5][7] The presence of ADP causes a conformational shift in GS that stabilizes the γ-glutamyl phosphate moiety. Ammonium binds strongly to GS only if the acyl-phosphate intermediate is present. Ammonium, rather than ammonia, binds to GS because the binding site is polar and exposed to solvent.^[7] In the second step, deprotonation of ammonium allows ammonia to attack the intermediate from its nearby site to form glutamine.^[12] Phosphate leaves through the top of the active site, while glutamine leaves through the bottom (between two rings).^[13][7]

Biological function

GS is present predominantly in the brain, kidneys, and liver.

There seem to be three different classes of GS:[18]^[19][20]

• Class I enzymes (GSI) are specific to prokaryotes, and are oligomers of 12 identical subunits.^[21] The activity of GSI-type enzyme is controlled by the adenylation of a tyrosine residue. The adenylated enzyme is inactive.^[22]
• Class II enzymes (GSII) are found in
  decamer of identical subunits.^[10]PDB: 2OJW
  ​.

Plants have two or more isozymes of GSII, one of the isozymes is translocated into the chloroplast. Another form is cytosolic. The cytosolic GS gene translation is regulated by its 5' untranslated region (UTR), while its 3' UTR plays role in transcript turnover.^[23]

• Class III enzymes (GSIII) have, currently, only been found in
  Butyrivibrio fibrisolvens. It is a double-ringed dodecamer of identical chains.^[24]
  It is much larger (about 700 amino acids) than the GSI (450 to 470 amino acids) or GSII (350 to 420 amino acids) enzymes.

Inhibition of GS has largely focused on amino site ligands.[6] Other inhibitors are the result of glutamine metabolism: tryptophan, histidine, carbamoyl phosphate, glucosamine-6-phosphate, cytidine triphosphate (CTP), and adenosine monophosphate (AMP).^[5][8][27] Other inhibitors/regulators are glycine and alanine. Alanine, glycine, and serine bind to the glutamate substrate site. GDP, AMP, ADP bind to the ATP site.^[6] L-serine, L-alanine, and glycine bind to the site for L-glutamate in unadenylated GS. The four amino acids bind to the site by their common atoms, “the main chain” of amino acids.^[5] Glutamate is another product of glutamine metabolism; however, glutamate is a substrate for GS inhibiting it to act as a regulator to GS.2 Each inhibitor can reduce the activity of the enzyme; once all final glutamine metabolites are bound to GS, the activity of GS is almost completely inhibited.^[8] Many inhibitory input signals allows for fine tuning of GS by reflecting nitrogen levels in the organism.

Feedback regulation distinguishes the difference between two eukaryotic types of GS: brain and non-brain tissues. Non-brain GS responds to end-product feedback inhibition, while brain GS does not.^[6] High concentrations of glutamine-dependent metabolites should inhibit GS activity, while low concentrations should activate GS activity.^[6]

Inhibitors:

• Methionine sulfoximine (MSO): MSO is an inhibitor that binds to the glutamate site. Bound to GS, MSO is phosphorylated by ATP that results in an irreversible, non-covalent inhibition of GS. The S-isomer configuration is more inhibitory.^[6] Glutamate entry is blocked into the active site by a stabilization of the flexible loop in the active site by MSO.^[7]
• Phosphinothricin^[1](PPT, Glufosinate): Phosphinothricin is an inhibitor that binds to the glutamate site. Glufosinate is used as an herbicide. Glufosinate treated plants die due to a buildup of ammonia and a cessation of photosynthesis.^[10]
• Many synthetic inhibitors are available today.[6]

Research on E. coli revealed that GS is regulated through gene expression. The gene that encodes the GS subunit is designated glnA. Transcription of glnA is dependent on NR_I (a specific transcriptional enhancer). Active transcription occurs if NR_I is in its phosphorylated form, designated NR_I-P. Phosphorylation of NR_I is catalyzed by NR_II, a protein kinase. If NR_II is complexed with P_IIA then it will function as a phosphatase and NR_I-P is converted back to NR_I. In this case, transcription of glnA ceases.^[25]

GS is subject to completely different regulatory mechanisms in