Glucosylceramidase

 

Acid beta-glucosidase (GCase) is a 497 amino acid membrane-associated lysosomal exo-beta-glucosidase, whose defective activity leads to the Gaucher disease. GCase cleaves the beta-glucosidic linkage of its major, natural substrate, glucosylceramide, as well as synthetic beta-glucosides. The 497 amino acid mature glycoprotein is derived from 517- or 536- amino acid precursors containing leader sequences that are removed during transit through the endoplasmic reticulum membrane. Cotranslational glycosylation occurs at four out of the five N-glycosylation sites. This glycosylation is essential for the development of the catalytically active enzyme. The catalytic cycle of GCase is dependent on the conformational changes during the transition state. Binding of effectors to specific sites of the enzyme induce the necessary changes required to active the enzyme.

 

Reference Protein and Structure

Sequence
P04062 UniProt (2.4.1.-, 3.2.1.-, 3.2.1.45) IPR001139 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
2f61 - Crystal structure of partially deglycosylated acid beta-glucosidase (2.5 Å) PDBe PDBsum 2f61
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 2f61)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.45)

beta-D-glucosyl-N-acylsphingosine
CHEBI:22801ChEBI
+
water
CHEBI:15377ChEBI
→
D-glucopyranose
CHEBI:4167ChEBI
+
N-acylsphingosine
CHEBI:52639ChEBI
Alternative enzyme names: Beta-D-glucocerebrosidase, Beta-glucosylceramidase, GlcCer-beta-glucosidase, Ceramide glucosidase, Glucocerebrosidase, Glucosphingosine glucosylhydrolase, Glucosylcerebrosidase, Glucosylsphingosine beta-D-glucosidase, Glucosylsphingosine beta-glucosidase, Psychosine hydrolase, Beta-glucocerebrosidase, Acid beta-glucosidase,

Enzyme Mechanism

Introduction

The mechanism begins with Glu274 acting as an acid protonating the glycosidic bond, this promotes its cleavage. this leads to the formation of an oxy-carbonium ion which can be attacked by the nucleophile Glu379, forming an enzyme-substrate intermediate. Glu274 then acts as a base activating a water molecule which hydrolyses the intermediate.

Catalytic Residues Roles

UniProt PDB* (2f61)
Glu274 Glu235B Glu 274 protonates the substrate O-glycosidic bond, activating it towards nucleophilic attack by Gluy 379. Deprotonation of a water molecule by an acid/base reaction liberates the hydroxyl ion, which is then able to attack the enzyme-glucose complex proton acceptor, proton donor, activator, increase nucleophilicity, promote heterolysis
Glu379 Glu340B Glu 340 attacks the substrate O-glycosidic bond, which is susceptible to nucleopphilic attack due to protonation from an acid/base reaction with Glu 235. covalently attached, nucleofuge, nucleophile
Cys381 Cys342B Cys 381 is located near to the active site nucleophile, Glu 379, and has significant conformational and local effects. This ensures that other residues are positioned appropriately for catalytic activity 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 product formed, overall reactant used, heterolysis, bimolecular nucleophilic addition, intermediate formation, native state of enzyme regenerated, hydrolysis, intermediate terminated, bimolecular nucleophilic substitution

References

  1. Liou B et al. (2006), J Biol Chem, 281, 4242-4253. Analyses of Variant Acid beta-Glucosidases: EFFECTS OF GAUCHER DISEASE MUTATIONS. DOI:10.1074/jbc.m511110200. PMID:16293621.
  2. Liou B et al. (2009), Mol Genet Metab, 97, 65-74. Participation of asparagine 370 and glutamine 235 in the catalysis by acid beta-glucosidase: the enzyme deficient in Gaucher disease. DOI:10.1016/j.ymgme.2009.01.006. PMID:19217815.
  3. Grace ME et al. (1994), J Biol Chem, 269, 2283-2291. Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression. PMID:8294487.
  4. Fabbro D et al. (1991), J Biol Chem, 266, 15021-15027. Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites. PMID:1714449.

Step 1. The glycosidic linkage is cleaved upon protonation by Glu274 and an oxy-carbonium ion is formed.

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Catalytic Residues Roles

Residue Roles
Glu235B promote heterolysis, proton donor

Chemical Components

proton transfer, overall product formed, overall reactant used, heterolysis

Step 2. Glu379 attacks the oxy-carbonium ion forming an enzyme-substrate intermediate.

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Catalytic Residues Roles

Residue Roles
Glu340B covalently attached
Cys342B electrostatic stabiliser
Glu340B nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 3. Glu274 activates a water molecule which hydrolyses the intermediate.

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Catalytic Residues Roles

Residue Roles
Glu235B activator, increase nucleophilicity
Glu340B nucleofuge
Glu235B proton acceptor

Chemical Components

native state of enzyme regenerated, hydrolysis, intermediate terminated, ingold: bimolecular nucleophilic substitution, proton transfer, overall product formed
Download: Image, Marvin File

Step 1. The glycosidic linkage is cleaved upon protonation by Glu274 and an oxy-carbonium ion is formed.

Step 2. Glu379 attacks the oxy-carbonium ion forming an enzyme-substrate intermediate.

Step 3. Glu274 activates a water molecule which hydrolyses the intermediate.

Products.

Contributors

Emma Penn, Gemma L. Holliday, James Willey