Mechanism and Catalytic Site Atlas

Golgi 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.