Gamma-glutamyl hydrolase

 

gamma-glutamyl hydrolase is a lysosomal or secreted thiol-dependent peptidase most active at acidic pH that catalyses the cleavage of the gamma-glutamyl chain of folylpoly-gamma-glutamyl. Human gamma-glutamyl hydrolase (hGH) catalyses the hydrolysis of the gamma-linked polyglutamate chain of folyl- or antifolyl polyglutamates, releasing shorter chains of polyglutamates and diglutamates or glutamic acid.

 

Reference Protein and Structure

Sequence
Q92820 UniProt (3.4.19.9) IPR015527 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1l9x - Structure of gamma-Glutamyl Hydrolase (1.6 Å) PDBe PDBsum 1l9x
Catalytic CATH Domains
3.40.50.880 CATHdb (see all for 1l9x)
Click To Show Structure

Enzyme Reaction (EC:3.4.19.9)

tetrahydrofolyl-poly(glutamic acid) macromolecule
CHEBI:28624ChEBI
+
water
CHEBI:15377ChEBI
→
5,6,7,8-tetrahydrofolic acid
CHEBI:20506ChEBI
+
L-glutamic acid
CHEBI:16015ChEBI
Alternative enzyme names: Gamma-Glu-X carboxypeptidase, Carboxypeptidase G, Conjugase, Folate conjugase, Folic acid conjugase, Lysosomal gamma-glutamyl carboxypeptidase, Poly(gamma-glutamic acid) endohydrolase, Poly(glutamic acid) hydrolase II, Polyglutamate hydrolase, Pteroyl-poly-gamma-glutamate hydrolase, Pteroylpoly-gamma-glutamyl hydrolase, Pteroyl-poly-alpha-glutamate hydrolase, GGH (gene name), Gamma-glutamyl hydrolase,

Enzyme Mechanism

Introduction

His220 activates Cys110 by abstracting a proton. The nucleophilic Cys110 attacks the gamma-carbonyl carbon of the of the susceptible Glu-Glu bond in the poly-gamma-glutamate substrate, forming a tetrahedral intermediate with an oxyanion. The carbonyl C-N bond is cleaved, forming the thioester intermediate and the gamma-linked glutamate leaving group which is protonated by His220. The thioester intermediate is hydrolysed by a water molecule which is activated by His220 acting as a base, yielding the product and regenerating the enzyme.

Catalytic Residues Roles

UniProt PDB* (1l9x)
Glu246 Glu222(243)B Raises the pKa of His 244 which enables the proton transfer between His 244 and Cys 132 electrostatic stabiliser
Cys134 Cys110(131)B Activated Cys110 nucleophilically attacks the gamma-carbonyl carbon of the substrate to form the thioester intermediate. nucleofuge, nucleophile, proton acceptor, proton donor
His244 His220(241)B His220 acts as a general acid/base catalyst. It activates the two nucleophiles in the reaction (Cys110 and water) as a base, and protonates the leaving group. 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, intermediate formation, overall reactant used, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Chave KJ et al. (2000), J Biol Chem, 275, 40365-40370. Molecular Modeling and Site-directed Mutagenesis Define the Catalytic Motif in Human gamma -Glutamyl Hydrolase. DOI:10.1074/jbc.m007908200. PMID:11005824.
  2. Alexander JP et al. (2008), Biochemistry, 47, 1228-1239. Gamma-glutamyl hydrolase: kinetic characterization of isopeptide hydrolysis using fluorogenic substrates. DOI:10.1021/bi701607v. PMID:18171026.
  3. Schneider E et al. (2006), Clin Chim Acta, 374, 25-32. Gamma-glutamyl hydrolase and drug resistance. DOI:10.1016/j.cca.2006.05.044. PMID:16859665.
  4. Li H et al. (2002), J Biol Chem, 277, 24522-24529. Three-dimensional structure of human gamma -glutamyl hydrolase. A class I glatamine amidotransferase adapted for a complex substate. DOI:10.1074/jbc.M202020200. PMID:11953431.
  5. Chave KJ et al. (1999), Biochem J, 343, 551-555. Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human γ-glutamyl hydrolase. DOI:10.1042/bj3430551. PMID:10527932.

Step 1. His244 deprotonates Cys134 which activates it so it can attack the carbon of the peptide bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu222(243)B electrostatic stabiliser
His220(241)B proton acceptor
Cys110(131)B proton donor, nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used, rate-determining step

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then accepts a proton from His244.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu222(243)B electrostatic stabiliser
His220(241)B proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, overall product formed

Step 3. His244 abstracts a proton from a water which activates it so it can attack the carbon of the thioester bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu222(243)B electrostatic stabiliser
His220(241)B proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 4. The oxyanion initiates another elimination which results in the cleavage of the thioester bond. The released Cys134 can now accept a proton from His244 which returns the enzyme to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu222(243)B electrostatic stabiliser
Cys110(131)B proton acceptor
His220(241)B proton donor
Cys110(131)B nucleofuge

Chemical Components

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

Step 1. His244 deprotonates Cys134 which activates it so it can attack the carbon of the peptide bond in a nucleophilic addition.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then accepts a proton from His244.

Step 3. His244 abstracts a proton from a water which activates it so it can attack the carbon of the thioester bond in a nucleophilic addition.

Step 4. The oxyanion initiates another elimination which results in the cleavage of the thioester bond. The released Cys134 can now accept a proton from His244 which returns the enzyme to its native state.

Products.

Contributors

Gemma L. Holliday, Charity Hornby