Glutamine synthetase

Class of enzymes

Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine:

Glutamate + ATP + NH3 → Glutamine + ADP + phosphate

Glutamine synthetase uses ammonia produced by nitrate reduction, amino acid degradation, and photorespiration. The amide group of glutamate is a nitrogen source for the synthesis of glutamine pathway metabolites. 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. Ammonium ion binds more strongly than water to GS due to electrostatic forces between a cation and a negatively charged pocket. Bacterial GS are dodecamers with 12 active sites between each monomer. ATP binds to the top of the bifunnel that opens to the external surface of GS. Phosphate leaves through the top of the active site, while glutamine leaves through the bottom (between two rings). GS, in the brain, is found primarily in astrocytes. Astrocytes protect neurons against excitotoxicity by taking up excess ammonia and glutamate. Different perspectives have approached the problem of astroglial swelling. One study shows that morphological changes occur that increase GS expression in glutamatergic areas or other adaptations that alleviates high levels of glutamate and ammonia. Class I enzymes (GSI) are specific to prokaryotes, and are oligomers of 12 identical subunits. The activity of GSI type enzyme is controlled by the adenylation of a tyrosine residue. The adenylated enzyme is inactive. Class II enzymes (GSII) are found in eukaryotes and in bacteria belonging to the Rhizobiaceae, Frankiaceae, and Streptomycetaceae families (these bacteria have also a class I GS). GSII are decamer of identical subunits. Class III enzymes (GSIII) have, currently, only been found in Bacteroides fragilis and in Butyrivibrio fibrisolvens. It is a double ringed dodecamer of identical chains. It is much larger (about 700 amino acids) than the GSI (450 to 470 amino acids) or GSII (350 to 420 amino acids) enzymes. While the three classes of GSs are clearly structurally related, the sequence similarities are not so extensive.

GS is subject to reversible covalent modification. Tyr397 of all 12 subunits can undergo adenylylation or deadenylylation by adenylyl transferase (AT), a bifunctional regulatory enzyme. Adenylylation is a post translational modification involving the covalent attachment of AMP to a protein side chain. Each adenylylation requires an ATP and complete inhibition of GS requires 12 ATP. Deadenylylation by AT involves phosphorolytic removal of the Tyr linked adenylyl groups as ADP. AT activity is influenced by the regulatory protein that is associated with it: PII, a 44 kD trimer. Inhibition of GS has largely focused on amino site ligands. 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. Instead of the common NtrC NtrB two component system, cyanobacteria harbour the transcriptional regulator NtcA which is restricted to this clade and controls expression of GS and a multitude of genes involved in nitrogen metabolism. Moreover, GS in cyanobacteria is not covalently modified to raise sensitivity for feedback inhibition. These inactivating factors are furthermore regulated by different Non coding RNAs: The sRNA NsiR4 interacts with the 5'UTR of the mRNA of the GS inactivating factor IF7 (gifA mRNA) and reduces its expression. NsiR4 expression is under positive control of the nitrogen control transcription factor NtcA. In addition, expression of the GS inactivating factor IF17 is controlled by a glutamine binding riboswitch.