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Catalytic Site Atlas Version 2.2.12
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CSA entry for 2f61
Original Entry
Title:
Hydrolase
Compound:
Acid beta-glucosidase
Mutant:
No
UniProt/Swiss-Prot:
P04062-GLCM_HUMAN
EC Class:
3.2.1.45
Other CSA Entries:
Overview of all sites for 2f61
Homologues of 2f61
Entries for UniProt/Swiss-Prot: P04062
Entries for EC: 3.2.1.45
Other Databases:
PDB entry: 2f61
PDBsum entry: 2f61
UniProt/Swiss-Prot: P04062
IntEnz entry: 3.2.1.45
Literature Report:
Introduction:
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 neccessary changes required to active the enzyme.
Mechanism:
The catalytic cycle proceeds through a two-step reaction mechanism. Glycosylation of the active site by the substrate is followed by deglycosylation with release of beta-glucose.

1. The substrate O-glycosidic bond is protonated by Glu 235.
2. Nucleophilic Glu 340 attacks the protonated glycosidic substrate bond.
3. Glucose becomes covalently bonded and the leaving group is removed.
4. Deprotonation of water by Glu 235.
5. The activated hydroxyl ion nucleophile attacks the enzyme-substrate complex, releasing beta-glucose.
Sites:

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLUA 235 274Sidechain
Acid/baseSubstrate
Acid/baseWater
Glu 235 protonates the substrate O-glycosidic bond, activating it towards nucleophilic attack by Gluy 340. Deprotonation of a water molecule by an acid/base reaction liberates the hydroxyl ion, which is then able to attack the enzyme-glucose complex
Evidence from paper Evidence concerns Evidence type
PubMed ID 16293621 Current protein Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLUA 340 379Sidechain
NucleophileSubstrate
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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 16293621 Current protein Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 342 381Sidechain
ElectrostaticTransition state
Cys 342 is located near to the active site nucleophile, Glu 340, and has significant conformational and local effects. This ensures that other residues are positioned appropriately for catalytic activity
Evidence from paper Evidence concerns Evidence type
PubMed ID 16293621 Current protein Mutagenesis of residue
Notes:
Further residues are thought to have indirectly catalytic roles. Arg 120 and Asn 370 are believed to have indirect roles by maintaining an active conformation and by altering active site access.

Cysteines involved in disulphide formation are essential to formation and/or preservation of an active enzyme.
References:
1
Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations.
B. Liou and A. Kazimierczuk and M. Zhang and C. R. Scott and R. S. Hegde and G. A. Grabowski
J Biol Chem 281, (7) 4242-53, (2006).
16293621
2
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.
D. Fabbro and G. A. Grabowski
J Biol Chem 266, (23) 15021-7, (1991).
1714449
3
Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression.
M. E. Grace and K. M. Newman and V. Scheinker and A. Berg-Fussman and G. A. Grabowski
J Biol Chem 269, (3) 2283-91, (1994).
8294487
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