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CSA entry for 1ex1
Original Entry
Title:
Hydrolase
Compound:
Beta-d-glucan exohydrolase isoenzyme exo1
Mutant:
No
UniProt/Swiss-Prot:
Q9XEI3-Q9XEI3
EC Class:
3.2.1.58
Other CSA Entries:
Overview of all sites for 1ex1
Homologues of 1ex1
Entries for UniProt/Swiss-Prot: Q9XEI3
Entries for EC: 3.2.1.58
Other Databases:
PDB entry: 1ex1
PDBsum entry: 1ex1
UniProt/Swiss-Prot: Q9XEI3
IntEnz entry: 3.2.1.58
Literature Report:
Introduction:
Barley beta-D-glucan exohydrolasesbelong to family 3 of glucosyl hydrolases. ExoI is one of two beta-D-glucan exohydrolase isoenzymes that hydrolyse a broad range of substrates with (1-2)-, (1-3)-, (1-4)-, and (1-6)-beta-D-glucosidic linkages. The enzyme removes single glucose units from the non-reducing termini of polymeric and oligomeric substrates with the retention of anomeric configuration.

The selective hydrolysis of glycosidic bonds is crucial for key processes of plant development and growth, such as cell wall expansion and degradation, and turnover of signalling molecules.

Mechanism:
The reaction follows a double displacement mechanism at the anomeric chiral centre.

Hydrolysis of the glycosidic linkage is believed to be initiated by protonation of the glycosidic oxygen atom via proton transfer from an active site-located general acid catalyst (Glu491) to form an oxonium ion.

Following protonation, the aglycone part of the substrate is released from the enzyme surface and the reducing terminal residue is converted to a positively charged oxycarbenium ion. The carbocation is stabilised by a nucleophilic amino acid residue (Asp285) acting as a base to form a covalent glucosyl-enzyme intermediate.

Glu491, acting as a base catalyst by proton abstraction, activates a water molecule which hydrolyses the covalent intermediate, resulting in the regeneration of the enzyme and the formation of the glucose product. The glucose remains bound to the enzyme until a new substrate approaches the enzyme.

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 285 310Sidechain
ElectrostaticTransition state
Acid/baseSubstrate
Asp285 is called a catalytic nucleophile, but acts as a base to form a covalent glucosyl-enzyme intermediate.It may also be involved in stabilising the carbocation transition state.
Evidence from paper Evidence concerns Evidence type
PubMed ID 10368285 Current protein pH dependence of reaction
PubMed ID 10368285 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 11709165 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLUA 491 516Sidechain
Acid/baseSubstrate
Acid/baseWater
Glu491 acts as a general acid/base catalyst. It protonates the substrate to initiate the hydrolysis mechanism. As a base, it activates water as a nucleophile to hydrolyse the covalent glycosyl-enzyme intermediate.
Evidence from paper Evidence concerns Evidence type
PubMed ID 10368285 Current protein pH dependence of reaction
PubMed ID 10368285 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 11709165 Current protein Residue is positioned appropriately (ligand position known)
Notes:
pH dependence data is in another article which does not have a PMID and is not on pubmed.

References:
Which EBI biological databases are available and how do I access them? EBI Site Map