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J. Biol. Chem., Vol. 280, Issue 27, 25313-25322, July 8, 2005

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O-GlcNAcase Uses Substrate-assisted Catalysis

KINETIC ANALYSIS AND DEVELOPMENT OF HIGHLY SELECTIVE MECHANISM-INSPIRED INHIBITORS^*

Matthew S. Macauley, Garrett E. Whitworth, Aleksandra W. Debowski, Danielle Chin, and David J. Vocadlo

From the Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

The post-translational modification of serine and threonine residues of nucleocytoplasmic proteins with 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) is a reversible process implicated in multiple cellular processes. The enzyme O-GlcNAcase catalyzes the cleavage of -O-linked GlcNAc (O-GlcNAc) from modified proteins and is a member of the family 84 glycoside hydrolases. The family 20 -hexosaminidases bear no apparent sequence similarity yet are functionally related to O-GlcNAcase because both enzymes cleave terminal GlcNAc residues from glycoconjugates. Lysosomal -hexosaminidase is known to use substrate-assisted catalysis involving the 2-acetamido group of the substrate; however, the catalytic mechanism of human O-GlcNAcase is unknown. By using a series of 4-methylumbelliferyl 2-deoxy-2-N-fluoroacetyl--D-glucopyranoside substrates, Taft-like linear free energy analyses of these enzymes indicates that O-GlcNAcase uses a catalytic mechanism involving anchimeric assistance. Consistent with this proposal, 1,2-dideoxy-2'-methyl--D-glucopyranoso-[2,1-d]-2'-thiazoline, an inhibitor that mimics the oxazoline intermediate proposed in the catalytic mechanism of family 20 glycoside hydrolases, is shown to act as a potent competitive inhibitor of both O-GlcNAcase (K_II = 0.070 µM) and -hexosaminidase (K = 0.070 µM). A series of 1,2-dideoxy-2'-methyl--D-glucopyranoso-[2,1-d]-2'-thiazoline analogues were prepared, and one inhibitor demonstrated a remarkable 1500-fold selectivity for O-GlcNAcase (K_I = 0.230 µM) over -hexosaminidase (K_I = 340 µM). These inhibitors are cell permeable and modulate the activity of O-GlcNAcase in tissue culture. Because both enzymes have vital roles in organismal health, these potent and selective inhibitors of O-GlcNAcase should prove useful in studying the role of this enzyme at the organismal level without generating a complex chemical phenotype stemming from concomitant inhibition of -hexosaminidase.

Received for publication, December 8, 2004 , and in revised form, March 3, 2005.

^* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Protein Engineering Networks of Centres of Excellence Research Chairs Program, starting funds from Simon Fraser University, and a President's Research Grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

Recipient of a Tier II Research Chair from the Canada Research Chairs Program. To whom correspondence should be addressed: Dept. of Chemistry, Simon Fraser University, 8888 University Blvd., Burnaby, British Columbia V5A 1S6, Canada. E-mail: dvocadlo{at}sfu.ca.

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