Glutaminase

 

Glutaminases (EC:3.5.1.2) deaminate glutamine to glutamate. In Bacillus subtilis, glutaminase is encoded by glnA, which is part of an operon, glnA-glnT (formerly ybgJ-ybgH), where glnT encodes a glutamine transporter. The glnA-glnT operon is regulated by the 2-component system GlnK-GlnL in response to glutamine [PMID:15995196].

 

Reference Protein and Structure

Sequence
O31465 UniProt (3.5.1.2) IPR015868 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis subsp. subtilis str. 168 (Bacteria) Uniprot
PDB
1mki - Crystal Structure of Bacillus Subtilis Probable Glutaminase, APC1040 (2.0 Å) PDBe PDBsum 1mki
Catalytic CATH Domains
1.10.1500.10 CATHdb 3.40.710.10 CATHdb (see all for 1mki)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.2)

L-glutamine zwitterion
CHEBI:58359ChEBI
+
water
CHEBI:15377ChEBI
→
L-glutamate(1-)
CHEBI:29985ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: L-glutaminase, Glutaminase I, Glutamine aminohydrolase, L-glutamine amidohydrolase,

Enzyme Mechanism

Introduction

Lys77 deprotonates Ser74, which initiates a nucleophilic addition onto the carbonyl carbon of the substrate amide group, forming a tetrahedral intermediate. The tetrahedral intermediate collapses, cleaving the C-N bond and releasing the ammonia product, the nitrogen of which deprotonates Lys77 via a proton relay through Tyr253. Lys77 deprotonates water via a proton relay with Tyr201, which initiates a nucleophilic addition at the carbonyl carbon, forming a new tetrahedral intermediate. The tetrahedral intermediate collapses, cleaving the acyl-enzyme bond and liberating Ser74, which in turn deprotonates the Lys77 via a proton relay with Tyr201.

Catalytic Residues Roles

UniProt PDB* (1mki)
Ser74 Ser74(77)A Acts as a nucleophile, forming a covalently attached intermediate. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, activator
Ser74 (main-N), Val271 (main-N) Ser74(77)A (main-N), Val271(274)A (main-N) Form the oxyanion hole that stabilises the negatively charged intermediates and transition states. hydrogen bond donor, electrostatic stabiliser
Tyr201 Tyr201(204)A Part of a proton relay chain (with Lys77) responsible for protonating the ammonia leaving group. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Tyr253 Tyr253(256)A Part of a proton relay chain (with Lys77) responsible for activating the catalytic water molecule. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay, activator, electrostatic stabiliser
Lys77 Lys77(80)A Acts as a general acid/base, responsible for activating the catalytic serine. hydrogen bond acceptor, hydrogen bond donor, 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, bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, proton relay, native state of enzyme regenerated, intermediate collapse, intermediate terminated

References

  1. Brown G et al. (2008), Biochemistry, 47, 5724-5735. Functional and Structural Characterization of Four Glutaminases from Escherichia coli and Bacillus subtilis†. DOI:10.1021/bi800097h. PMID:18459799.

Step 1. Lys77 deprotonates Ser74, which initiates a nucleophilic addition onto the carbonyl carbon of the substrate amide group, forming a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val271(274)A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser74(77)A (main-N) hydrogen bond donor, electrostatic stabiliser
Tyr253(256)A hydrogen bond acceptor, activator, hydrogen bond donor, electrostatic stabiliser
Tyr201(204)A hydrogen bond acceptor
Lys77(80)A hydrogen bond acceptor, proton acceptor
Ser74(77)A proton donor, nucleophile

Chemical Components

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

Step 2. The tetrahedral intermediate collapses, cleaving the C-N bond and releasing the ammonia product, the nitrogen of which deprotonates Lys77 via a proton relay through Tyr253.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val271(274)A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser74(77)A (main-N) hydrogen bond donor, electrostatic stabiliser
Tyr253(256)A hydrogen bond acceptor, hydrogen bond donor, proton relay
Tyr201(204)A hydrogen bond acceptor
Lys77(80)A hydrogen bond donor
Ser74(77)A covalently attached, activator
Lys77(80)A proton donor
Tyr253(256)A proton acceptor, proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, intermediate formation, proton relay

Step 3. Lys77 deprotonates water via a proton relay with Tyr201, which initiates a nucleophilic addition at the carbonyl carbon, forming a new tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val271(274)A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser74(77)A (main-N) hydrogen bond donor, electrostatic stabiliser
Tyr253(256)A hydrogen bond acceptor
Tyr201(204)A hydrogen bond acceptor, hydrogen bond donor, proton relay
Lys77(80)A hydrogen bond acceptor
Ser74(77)A activator, covalently attached
Tyr201(204)A proton acceptor, proton donor
Lys77(80)A proton acceptor

Chemical Components

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

Step 4. The tetrahedral intermediate collapses, cleaving the acyl-enzyme bond and liberating Ser74, which in turn deprotonates the Lys77 via a proton relay with Tyr201.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val271(274)A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser74(77)A (main-N) hydrogen bond donor, electrostatic stabiliser
Tyr253(256)A hydrogen bond acceptor
Tyr201(204)A hydrogen bond acceptor, hydrogen bond donor, proton relay
Lys77(80)A hydrogen bond donor
Tyr201(204)A proton acceptor
Ser74(77)A proton acceptor
Lys77(80)A proton donor
Ser74(77)A nucleofuge
Tyr201(204)A proton donor

Chemical Components

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

Step 1. Lys77 deprotonates Ser74, which initiates a nucleophilic addition onto the carbonyl carbon of the substrate amide group, forming a tetrahedral intermediate.

Step 2. The tetrahedral intermediate collapses, cleaving the C-N bond and releasing the ammonia product, the nitrogen of which deprotonates Lys77 via a proton relay through Tyr253.

Step 3. Lys77 deprotonates water via a proton relay with Tyr201, which initiates a nucleophilic addition at the carbonyl carbon, forming a new tetrahedral intermediate.

Step 4. The tetrahedral intermediate collapses, cleaving the acyl-enzyme bond and liberating Ser74, which in turn deprotonates the Lys77 via a proton relay with Tyr201.

Products.

Contributors

Gemma L. Holliday