Glutamate dehydrogenase

 

Glutamate dehydrogenases catalyse the reversible oxidative deamination of L-glutamate, with NAD(P)H as cofactor. Glutamatc dehydrogcnases arc widely distributed throughout the cukaryotic, cubacterial and arc-haebaoterial kingdoms and these enzymes form a major link between protein and carbohydrate metabolism.

 

Reference Protein and Structure

Sequence
P24295 UniProt (1.4.1.2) IPR014362 (Sequence Homologues) (PDB Homologues)
Biological species
[Clostridium] symbiosum (Bacteria) Uniprot
PDB
1hrd - GLUTAMATE DEHYDROGENASE (1.96 Å) PDBe PDBsum 1hrd
Catalytic CATH Domains
3.40.50.10860 CATHdb (see all for 1hrd)
Click To Show Structure

Enzyme Reaction (EC:1.4.1.2)

L-glutamate(1-)
CHEBI:29985ChEBI
+
water
CHEBI:15377ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
→
2-oxoglutarate(2-)
CHEBI:16810ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: L-glutamate dehydrogenase, NAD-dependent glutamate dehydrogenase, NAD-dependent glutamic dehydrogenase, NAD-glutamate dehydrogenase, NAD-linked glutamate dehydrogenase, NAD-linked glutamic dehydrogenase, NAD-specific glutamate dehydrogenase, NAD-specific glutamic dehydrogenase, NAD:glutamate oxidoreductase, NADH-linked glutamate dehydrogenase, Glutamate dehydrogenase (NAD), Glutamate oxidoreductase, Glutamic acid dehydrogenase, Glutamic dehydrogenase,

Enzyme Mechanism

Introduction

The first step in the mechanism is the deprotonation of the alpha-amino group of the glutamate by Asp165, acting as general base, which occurs with subsequent hydride transfer to the Si face of the NAD+ and leads to the production of an iminoglutarate intermediate. The next step is the attack of a water molecule on the iminoglutarate intermediate is enhanced byLys125 acting as a general base via a hydrogen bond. During the generation of the carbinolamine intermediate and its subsequent collapse to the 2-oxoacid, Asp165 is crucial for the transfer of the proton to and from the substrate. Finally the loss of a proton from Lys125 and Asp165 ends the catalytic cycle.

Catalytic Residues Roles

UniProt PDB* (1hrd)
Lys126 Lys125A Low pKa of residue enable it to act as a hydrogen-bond acceptor to enhance the nucleophilicity of a water moelcule to enable attack on the iminoglutarate intermediate. proton acceptor, electrostatic stabiliser, proton donor
Asp166 Asp165A General base in the initial deprotonation of the alpha-amino group of the glutamate. Acid/base catalyst during generation of 2-oxoacid intermediate and collapse. proton acceptor, proton donor
*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, aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, hydride transfer, bimolecular nucleophilic addition, intermediate formation, overall product formed, elimination (not covered by the Ingold mechanisms), deamination, inferred reaction step

References

  1. Dean JL et al. (1994), Biochem J, 301, 13-16. The catalytic role of aspartate in the active site of glutamate dehydrogenase. DOI:10.1042/bj3010013. PMID:8037659.
  2. Stillman TJ et al. (1993), J Mol Biol, 234, 1131-1139. Conformational Flexibility in Glutamate Dehydrogenase. DOI:10.1006/jmbi.1993.1665. PMID:8263917.

Step 1. Asp165 acts as a general base to deprotonate the amine group of the substrate. This occurs with subsequent hydride transfer to the Si face of NAD+ and leads to the formation of an iminoglutamate intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys125A electrostatic stabiliser
Asp165A proton acceptor

Chemical Components

proton transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, hydride transfer

Step 2. Lys125 activates a water molecule for nucleophilic attack on the imine intermediate. Asp165 transfers a proton to the developing amine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys125A proton acceptor
Asp165A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation

Step 3. Asp165 abstracts a proton from the hydroxyl group of the carbinolamine intermediate. The intermediate collapses and ammonia is eliminated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp165A proton acceptor

Chemical Components

proton transfer, overall product formed, elimination (not covered by the Ingold mechanisms), deamination

Step 4. In an inferred step Asp165 and Lys125 are deprotonated to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys125A proton donor
Asp165A proton donor

Chemical Components

inferred reaction step, proton transfer
Download: Image, Marvin File

Step 1. Asp165 acts as a general base to deprotonate the amine group of the substrate. This occurs with subsequent hydride transfer to the Si face of NAD+ and leads to the formation of an iminoglutamate intermediate.

Step 2. Lys125 activates a water molecule for nucleophilic attack on the imine intermediate. Asp165 transfers a proton to the developing amine.

Step 3. Asp165 abstracts a proton from the hydroxyl group of the carbinolamine intermediate. The intermediate collapses and ammonia is eliminated.

Step 4. In an inferred step Asp165 and Lys125 are deprotonated to regenerate the active site.

Products.

Contributors

Anna Waters, Craig Porter, Gemma L. Holliday, Amelia Brasnett