Resource type
Date created
2006-02-24
Authors/Contributors
Author (aut): Whitworth, Garrett E.
Author (aut): Macauley, Matthew S.
Author (aut): Stubbs, Keith A.
Author (aut): Dennis, Rebecca J.
Author (aut): Taylor, Edward J.
Author (aut): Davies, Gideon J.
Author (aut): Greig, Ian R.
Author (aut): Vocadlo, David J.
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.
Document
Identifier
DOI: 10.1021/bi052370b
Published as
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.
Publication details
Publication title
Biochemistry
Document title
Identification of Asp174 and Asp175 as the Key Catalytic Residues of Human O-GlcNAcase by Functional Analysis of Site-Directed Mutants
Date
2006
Volume
45
Issue
11
First page
3835
Last page
3844
Published article URL
Copyright statement
Copyright is held by the author(s).
Scholarly level
Peer reviewed?
Yes
Funder
Language
English
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