GMP synthase (glutamine-hydrolysing)

 

The Class I glutamine amidotransferase an example of a single enzymatic reaction being catalysed by two modules, each responsible for a distinct component of the reaction. It catalyses the amination of the nucleotide precursor xanthosine 5'-monophosphate to form GMP in the de novo purine biosynthesis pathway. The amidotransferase domain is found in related enzymes of the purine, pyrimidine, tryptophan, arginine, histidine and folic acid pathways. This domain includes a conserved Cys-His-Glu triad, responsible for the abstraction of the amide nitrogen from glutamine.

Despite the apparent catalytic readiness of the GMP synthetase active site, the Class I aminotransferase domain is actually a very poor glutaminase in the absence of the substrates XMP and ATP, which is consistent with the biological role of this enzyme, in which the hydrolysis of glutamine is tightly couple with the formation of GMP. The synthetase domain catalyses the addition of ammonia to an acceptor substrate. They are designed to work in concert to ensure efficient coupling of catalytic functions, it is suggested that a flexible hinge exists to bring the two sites together for concerted ammonia transfer.

 

Reference Protein and Structure

Sequence
P04079 UniProt (6.3.5.2) IPR022955 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1gpm - ESCHERICHIA COLI GMP SYNTHETASE COMPLEXED WITH AMP AND PYROPHOSPHATE (2.2 Å) PDBe PDBsum 1gpm
Catalytic CATH Domains
3.40.50.620 CATHdb 3.40.50.880 CATHdb (see all for 1gpm)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.5.2)

L-glutamine zwitterion
CHEBI:58359ChEBI
+
5'-xanthylate(2-)
CHEBI:57464ChEBI
+
water
CHEBI:15377ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
L-glutamate(1-)
CHEBI:29985ChEBI
+
hydron
CHEBI:15378ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
guanosine 5'-monophosphate(2-)
CHEBI:58115ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: GMP synthetase (glutamine-hydrolyzing), Guanosine 5'-monophosphate synthetase, Guanosine monophosphate synthetase (glutamine-hydrolyzing), Guanylate synthetase (glutamine-hydrolyzing), Xanthosine 5'-phosphate amidotransferase,

Enzyme Mechanism

Introduction

The beta-phosphate deprotonates the XMP substrate, which then initiates a nucleophilic attack on the alpha-phosphate of ATP, eliminating pyrophosphate aided by Asp239 and Lys381. His181 deprotonates Cys86, activating it for a nucleophilic attack upon L-glutamine, forming an enzyme-substrate covalent bond. The negative charge is stabilised, by an oxyanion hole involving Tyr87 and Gly59. The tetrahedral intermediate collapses, liberating ammonia, which deprotonates His181 and then passes to the other catalytic domain. Ammonia is deprotonated by the phosphate of the intermediate, activating it for a nucleophilic attack on the intermediate, which liberates AMP and the GMP product. In the glutamine site His181 deprotonates a water molecule, which initiates a nucleophilic attack on the Cys-bound intermediate. The tetrahedral intermediate collapses, liberating Cys86, which deprotonates His181, and the glutamate product.

Catalytic Residues Roles

UniProt PDB* (1gpm)
His181 His181A Part of the catalytic Glu-His-Cys triad. Acts as a general acid/base, activates the cysteine of the triad. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys381 Lys381A Helps to stabilise the build up of negative charge in the ATP-pyrophosphatase domain. hydrogen bond donor, electrostatic stabiliser
Cys86 Cys86A Part of the catalytic Glu-His-Cys triad. Acts as a general acid/base and as the catalytic nucleophile. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, nucleofuge, proton donor, proton acceptor
Glu183 Glu183A Part of the catalytic Glu-His-Cys triad. Responsible for stabilising and activating the His of the triad. increase basicity, hydrogen bond acceptor, increase acidity
Asp239, Asp239 Asp239A, Asp239A Forms part of the magnesium binding site. Also acts to stabilise and hold the reactants in the correct orientation for the reaction to occur. hydrogen bond acceptor, metal ligand, electrostatic stabiliser, steric role
Tyr87 (main-N), Gly59 (main-N) Tyr87A (main-N), Gly59A (main-N) Form the oxyanion hole that stabilises the negatively charged intermediates and transition states in the glutaminase domain. hydrogen bond donor, 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

bimolecular nucleophilic substitution, proton transfer, dephosphorylation, intermediate formation, overall product formed, overall reactant used, bimolecular nucleophilic addition, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, deamination, intermediate terminated, native state of enzyme regenerated

References

  1. Tesmer JJ et al. (1996), Nat Struct Biol, 3, 74-86. The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families. DOI:10.1038/nsb0196-74. PMID:8548458.
  2. Ballut L et al. (2015), Nat Commun, 6, 8930-. Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation. DOI:10.1038/ncomms9930. PMID:26592566.
  3. Massière F et al. (1998), Cell Mol Life Sci, 54, 205-222. The mechanism of glutamine-dependent amidotransferases. DOI:10.1007/s000180050145. PMID:9575335.

Step 1. The beta-phosphate deprotonates the XMP substrate, which then initiates a nucleophilic attack on the alpha-phosphate of ATP, eliminating pyrophosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys381A electrostatic stabiliser, hydrogen bond donor
Asp239A metal ligand, electrostatic stabiliser, steric role, hydrogen bond acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, dephosphorylation, intermediate formation, overall product formed, overall reactant used

Step 2. His181 deprotonates Cys86, activating it for a nucleophilic attack upon L-glutamine, forming an enzyme-substrate covalent bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly59A (main-N) electrostatic stabiliser, hydrogen bond donor
Cys86A hydrogen bond donor
His181A hydrogen bond acceptor, hydrogen bond donor
Glu183A increase basicity, hydrogen bond acceptor
Tyr87A (main-N) electrostatic stabiliser, hydrogen bond donor
His181A proton acceptor
Cys86A nucleophile, proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used

Step 3. The tetrahedral intermediate collapses, liberating ammonia, which deprotonates His181 and then passes to the other catalytic domain. The ammonia produced is transferred from the glutaminase domain to the ATP pyrophosphatase domain. However, the glutaminase and ATP pyrophosphatase domain are approximately 30 Angstroms apart, and it is not yet entirely clear how the liberated ammonia is transported from the glutaminase to the ATP pyrophosphatase domain. It has been suggested that the most likely route is by channelling through the protein in a manner analogous to the classical channelling of common metabolites between sequential enzyme pairs [PMID:9575335]. However, a hinging movement between the two domains has also been suggested [PMID:8548458].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly59A (main-N) electrostatic stabiliser, hydrogen bond donor
Cys86A covalently attached
His181A hydrogen bond donor
Glu183A hydrogen bond acceptor, increase acidity
Tyr87A (main-N) electrostatic stabiliser, hydrogen bond donor
His181A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, enzyme-substrate complex cleavage, deamination, intermediate formation

Step 4. Ammonia is deprotonated by the phosphate of the intermediate, activating it for a nucleophilic attack on the intermediate, which liberates AMP and the GMP product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys381A hydrogen bond donor
Asp239A metal ligand, hydrogen bond acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, intermediate terminated, intermediate collapse, overall product formed

Step 5. His181 deprotonates a water molecule, which initiates a nucleophilic attack on the Cys-bound intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly59A (main-N) hydrogen bond donor, electrostatic stabiliser
Cys86A covalently attached
His181A hydrogen bond donor, hydrogen bond acceptor
Glu183A hydrogen bond acceptor, increase basicity
Tyr87A (main-N) hydrogen bond donor, electrostatic stabiliser
His181A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 6. The tetrahedral intermediate collapses, liberating Cys86, which deprotonates His181, and the glutamate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly59A (main-N) hydrogen bond donor, electrostatic stabiliser
Cys86A covalently attached, hydrogen bond acceptor
His181A hydrogen bond donor
Glu183A hydrogen bond acceptor, increase acidity
Tyr87A (main-N) hydrogen bond donor, electrostatic stabiliser
His181A proton donor
Cys86A nucleofuge, proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The beta-phosphate deprotonates the XMP substrate, which then initiates a nucleophilic attack on the alpha-phosphate of ATP, eliminating pyrophosphate.

Step 2. His181 deprotonates Cys86, activating it for a nucleophilic attack upon L-glutamine, forming an enzyme-substrate covalent bond.

Step 3. The tetrahedral intermediate collapses, liberating ammonia, which deprotonates His181 and then passes to the other catalytic domain. The ammonia produced is transferred from the glutaminase domain to the ATP pyrophosphatase domain. However, the glutaminase and ATP pyrophosphatase domain are approximately 30 Angstroms apart, and it is not yet entirely clear how the liberated ammonia is transported from the glutaminase to the ATP pyrophosphatase domain. It has been suggested that the most likely route is by channelling through the protein in a manner analogous to the classical channelling of common metabolites between sequential enzyme pairs [PMID:9575335]. However, a hinging movement between the two domains has also been suggested [PMID:8548458].

Step 4. Ammonia is deprotonated by the phosphate of the intermediate, activating it for a nucleophilic attack on the intermediate, which liberates AMP and the GMP product.

Step 5. His181 deprotonates a water molecule, which initiates a nucleophilic attack on the Cys-bound intermediate.

Step 6. The tetrahedral intermediate collapses, liberating Cys86, which deprotonates His181, and the glutamate product.

Products.

Contributors

Gemma L. Holliday, Sophie T. Williams, Anna Waters, Craig Porter, Charity Hornby