Glutamine synthetase (type I)

 

Glutamine synthetase catalyses the condensation reaction of ammonium and glutamate to form glutamine, using ATP. This is observed in the early stages of infection by Mycobacterium tuberculosis as an extracellular component in the synthesis of poly(L-glutamine-L-glutamate) chains requires for cell wall formation. Therefore this enzyme is an important drug target for halting tuberculosis infection.

 

Reference Protein and Structure

Sequence
P9WN39 UniProt (6.3.1.2) IPR004809 (Sequence Homologues) (PDB Homologues)
Biological species
Mycobacterium tuberculosis H37Rv (Bacteria) Uniprot
PDB
2bvc - Crystal structure of Mycobacterium tuberculosis glutamine synthetase in complex with a transition state mimic (2.1 Å) PDBe PDBsum 2bvc
Catalytic CATH Domains
3.10.20.70 CATHdb 3.30.590.10 CATHdb (see all for 2bvc)
Cofactors
Magnesium(2+) (3)
Click To Show Structure

Enzyme Reaction (EC:6.3.1.2)

ammonium
CHEBI:28938ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
L-glutamate(1-)
CHEBI:29985ChEBI
→
hydrogenphosphate
CHEBI:43474ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
L-glutamine zwitterion
CHEBI:58359ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Glutamate--ammonia ligase, L-glutamine synthetase, Glutamylhydroxamic synthetase,

Enzyme Mechanism

Introduction

The reaction mechanism can be described as a series of loop and side-chain movements. ATP binds within the top of the bifunnel, its terminal phosphate group binding adjacent to an Mn ion. The binding of ATP results in loop movements. Glutamate enters the cavity and binds with its carboxylate group binding adjacent to a Mn ion or a Mg ion as these are interchangeable and do not affect the reaction mechanism and here the reaction is with Mg. Ammonium also enters the active site and is deprotonated by the side chain of Asp-50 to produce ammonia. The gamma-phosphate of ATP is transferred to the carboxylate of glutamate, thereby forming the intermediate. The two positively charged metal ions and Arg-339 and Arg-368 participate in phosphoryl transfer by polarizing the gamma-phosphate group of ATP making the phosphorus more positive. Ammonia attacks the N-carbon of the glutamyl phosphate intermediate, thereby releasing the phosphate group. The phosphate can deprotonate glutamine and the flap opens and glutamine is released.

Catalytic Residues Roles

UniProt PDB* (2bvc)
Asp54 Asp54(62)F Depronates the ammonium substrate ion. Increases the affinity for ammonium binding. activator, proton acceptor
Glu227, Glu133, Glu366, His276, Glu135, Glu219 Glu227(235)A, Glu133(141)A, Glu366(374)A, His276(284)A, Glu135(143)A, Glu219(227)A bind metal cofactor metal ligand
Arg368, Arg347 Arg368(376)A, Arg347(355)A Polarises the gamma-phosphate group of ATP making the phosphorus more positive. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, overall reactant used, intermediate formation, bimolecular nucleophilic addition, overall product formed, bimolecular nucleophilic substitution, rate-determining step, native state of enzyme is not regenerated

References

  1. Moreira C et al. (2016), Chemistry, 22, 9218-9225. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations. DOI:10.1002/chem.201600305. PMID:27225077.
  2. Moreira C et al. (2017), J Phys Chem B, 121, 6313-6320. Clarifying the Catalytic Mechanism of Human Glutamine Synthetase: A QM/MM Study. DOI:10.1021/acs.jpcb.7b02543. PMID:28587465.
  3. Krajewski WW et al. (2005), Proc Natl Acad Sci U S A, 102, 10499-10504. Structure of Mycobacterium tuberculosis glutamine synthetase in complex with a transition-state mimic provides functional insights. DOI:10.1073/pnas.0502248102. PMID:16027359.
  4. Gill HS et al. (2002), Biochemistry, 41, 9863-9872. Multicopy Crystallographic Refinement of a Relaxed Glutamine Synthetase fromMycobacterium tuberculosisHighlights Flexible Loops in the Enzymatic Mechanism and Its Regulation†. DOI:10.1021/bi020254s. PMID:12146952.
  5. Eisenberg D et al. (2000), Biochim Biophys Acta, 1477, 122-145. Structure–function relationships of glutamine synthetases. DOI:10.1016/s0167-4838(99)00270-8. PMID:10708854.
  6. Liaw SH et al. (1994), Biochemistry, 33, 675-681. Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes. DOI:10.1021/bi00169a007. PMID:7904828.
  7. Alibhai M et al. (1994), Biochemistry, 33, 682-686. Kinetic and mutagenic studies of the role of the active site residues Asp-50 and Glu-327 of Escherichia coli glutamine synthetase. DOI:10.1021/bi00169a008. PMID:7904829.

Step 1. Asp50 deprotonates the ammonium ion to produce ammonia. This activates ammonia.

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Catalytic Residues Roles

Residue Roles
Arg347(355)A electrostatic stabiliser
Arg368(376)A electrostatic stabiliser
Glu133(141)A metal ligand
Glu135(143)A metal ligand
Glu219(227)A metal ligand
Glu227(235)A metal ligand
His276(284)A metal ligand
Glu366(374)A metal ligand
Asp54(62)F activator, proton acceptor

Chemical Components

proton transfer, overall reactant used

Step 2. Substrate nucleophilically attacks Phosphorus on ATP resulting in the γ-phosphate being transferred to glutamate. This activates glutamate by increasing its electerophilicity.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu133(141)A metal ligand
Glu135(143)A metal ligand
Glu219(227)A metal ligand
Glu227(235)A metal ligand
His276(284)A metal ligand
Arg347(355)A electrostatic stabiliser
Glu366(374)A metal ligand
Arg368(376)A electrostatic stabiliser

Chemical Components

intermediate formation, overall reactant used, ingold: bimolecular nucleophilic addition, overall product formed

Step 3. Ammonia nucleophilically attacks the δ‐carbon of γ‐glutamyl phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu133(141)A metal ligand
Glu135(143)A metal ligand
Glu219(227)A metal ligand
Glu227(235)A metal ligand
His276(284)A metal ligand
Arg347(355)A electrostatic stabiliser
Glu366(374)A metal ligand
Arg368(376)A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution, rate-determining step

Step 4. Phosphate deprotonates Glutamine. The enzyme is not regenerated in this last step since Asp50 is still protonated. This proton will probably be released into the solution to return the enzyme to its original state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu133(141)A metal ligand
Glu135(143)A metal ligand
Glu219(227)A metal ligand
Glu227(235)A metal ligand
His276(284)A metal ligand
Arg347(355)A electrostatic stabiliser
Glu366(374)A metal ligand
Arg368(376)A electrostatic stabiliser

Chemical Components

proton transfer, overall product formed, native state of enzyme is not regenerated
Download: Image, Marvin File

Step 1. Asp50 deprotonates the ammonium ion to produce ammonia. This activates ammonia.

Step 2. Substrate nucleophilically attacks Phosphorus on ATP resulting in the γ-phosphate being transferred to glutamate. This activates glutamate by increasing its electerophilicity.

Step 3. Ammonia nucleophilically attacks the δ‐carbon of γ‐glutamyl phosphate.

Step 4. Phosphate deprotonates Glutamine. The enzyme is not regenerated in this last step since Asp50 is still protonated. This proton will probably be released into the solution to return the enzyme to its original state.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, Charity Hornby