Identification of Asp174 and Asp175 as the Key Catalytic Residues of Human O-GlcNAcase by Functional Analysis of Site-Directed Mutants

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Çetinbaş, N., Macauley, M. S., Stubbs, K. A., Drapala, R., & Vocadlo, D. J. (2006). Identification of Asp174 and Asp175 as the Key Catalytic Residues of Human O-GlcNAcase by Functional Analysis of Site-Directed Mutants. Biochemistry, 45(11), 3835–3844. https://doi.org/10.1021/bi052370b.

Date created: 
2006-02-24
Identifier: 
DOI: 10.1021/bi052370b
Keywords: 
Azides
Kinetic parameters
Peptides and proteins
Carbohydrates
Monomers
Abstract: 

O-GlcNAcase is a family 84 â-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of â-O-linked 2-acetamido-2-deoxy-D-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants of human O-GlcNAcase, D174A and D175A, were generated since these residues are highly conserved among family 84 glycoside hydrolases. Structure-reactivity studies of the D174A mutant enzyme reveals severely impaired catalytic activity across a broad range of substrates alongside a pH-activity profile consistent with deletion of a key catalytic residue. The D175A mutant enzyme shows a significant decrease in catalytic efficiency with substrates bearing poor leaving groups (up to 3000-fold), while for substates bearing good leading groups the difference is much smaller (7-fold). This mutant enzyme also cleaves thioglycosides with essentially the same catalytic efficiency as the wild-type enzyme. As well, addition of azide as an exogenous nucleophile increases the activity of this enzyme toward a substrate bearing an excellent leaving group. Together, these results allow unambiguous assignment of Asp174 as the residue that polarizes the 2-acetamido group for attack on the anomeric center and Asp175 as the residue that functions as the general acid/base catalyst. Therefore, the family 84 glycoside hydrolases use a DD catalytic pair to effect catalysis.

Language: 
English
Document type: 
Article
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Sponsor(s): 
Natural Sciences and Engineering Research Council of Canada (NSERC)
Protein Engineering Centers of Excellence (PENCE)
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