title: O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors.
abstract: 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 beta-O-linked GlcNAc (O-GlcNAc) from modified proteins and is a member of the family 84 glycoside hydrolases. The family 20 beta-hexosaminidases bear no apparent sequence similarity yet are functionally related to O-GlcNAcase because both enzymes cleave terminal GlcNAc residues from glycoconjugates. Lysosomal beta-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-beta-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-alpha-D-glucopyranoso-[2,1-d]-Delta2'-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(I) = 0.070 microm) and beta-hexosaminidase (K = 0.070 microm). A series of 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline analogues were prepared, and one inhibitor demonstrated a remarkable 1500-fold selectivity for O-GlcNAcase (K(I) = 0.230 microm) over beta-hexosaminidase (K(I) = 340 microm). 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 beta-hexosaminidase.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Macauley
authors.forename: Garrett E
authors.initials: GE
authors.lastname: Whitworth
authors.forename: Aleksandra W
authors.initials: AW
authors.lastname: Debowski
authors.forename: Danielle
authors.initials: D
authors.lastname: Chin
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000123:Acetyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology*; D006001:Glycoconjugates / Q000378:metabolism; D051548:Histone Acetyltransferases; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013379:Substrate Specificity; D013844:Thiazoles / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D006001:Glycoconjugates; D009097:Multienzyme Complexes; D013844:Thiazoles; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; C110628:hexosaminidase C; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; C487818:N-acetylglucosamine thiazoline; D000117:Acetylglucosamine