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Catalytic Site Atlas

CSA LITERATURE entry for 1qwn

E.C. namemannosyl-oligosaccharide 1,3-1,6-alpha-mannosidase
SpeciesDrosophila melanogaster (Fruit Fly)
E.C. Number (IntEnz) 3.2.1.114
CSA Homologues of 1qwn
CSA Entries With UniProtID Q24451
CSA Entries With EC Number 3.2.1.114
PDBe Entry 1qwn
PDBSum Entry 1qwn
MACiE Entry 1qwn

Literature Report

IntroductionGolgi alpha-mannosidase II (dGMII) from Drosophila melanogaster is a member of the glycosyl hydrolase family 38. It catalyses the hydrolysis of the terminal 1,3- and 1,6-linked alpha-D-mannose (Man) residues in the mannosyl-oligosaccharide Man(5)(GlcNAc)(3) producing Man(3)(GlcNAc)(3). GlcNAc stands for N-acetylglucosamine.
The enzyme has a high degree of conservation of sequence among many eukaryotes. Inhibition of the human GMII may be helpful in the treatmant of breast, colon or skin cancer.
GMII is involved in the N-linked glycosylation pathway which begins in the endoplasmic reticulum where an oligosaccharide is attached to an asparagine residue on a nascent polypeptide. GMII is located in the Golgi apparatus and is one of the glycosyl hydrolases involved in trimming the oligosaccharide.
The active site of GMII consitsis of a ctalytic site, a holding site, and an anchor site. Initially the alpha1,6-linked mannose residue of the substrate binds in the catalytic site while the alpha1,3-linked mannose bind in the holding site. A terminal N-acetylglucosamine residue of the substrate binds in the holding site and helps orient the substrate for the hydrolysis reaction. The holding site cannot accommodate the alpha1,6-linked residue without displacing the terminal N-acetylglucosamine from the anchor site. This ensures that the alpha1,6 bond is hydrolysed before the alpha1,3 bond. In in vitro studies dGMII shows an 80-fold preference for substrates containing a nonreducing beta(1,2)-linked GlcNAc which can occupy the anchor site. It has been proposed that the conformation of the catalytic site depends on the presence of a GlcNAc residue in the anchoring site.
MechansimThe two glycosidic bonds are hydrolysed in the same catalytic site. The bond to the alpha1,6-linked mannose residue is hydrolysed first. This is followed by a rearrangement of the substrate which results in the repositioning of the alpha1,3-linked mannose residue from the holding site into the catalytic site. The alpha1,3 bond is subsequently hydrolysed.
The hydrolysis of both the alpha1,6 and the alpha1,3 bonds occurs through a double displacement mechanism. First, a covalent glycosyl-enzyme intermediate is formed by the nucleophilic attack of the Asp204 side chain on the C1 position of the substrate. Second, the covalent glycosyl-enzyme intermediate is hydrolysed and the mannose is released.
As the covalent intermediate is formed the mannose residue which is attacked by the nucleophile (Asp204) addopts a 1S5 skew boat conformation. This is thought to minimise steric hindrance to the subsequent attack by the nucleophilic water and lower the energy barrier for transition state formation.
Reaction

Catalytic Sites for 1qwn

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
AspA204267macie:sideChainAsp204 functions as a nuclephile attacking the C1 position of the mannose residue in the catalytic site. This results in the formation of a glycosyl-enzyme intermediate.
AspA341404macie:sideChainAsp341 acts as a general acid catalyst in the first step of the hydrolysis by protonating the substrate as it is attacked by the nucleophile. In the second step, it deprotonates a water molecule to form a hydroxyl ion which preforms a nucleophilic attack on C1 of the saccharide in the glycosyl-enzyme intermediate.

Literature References

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