O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors

• Published in: The Journal of biological chemistry Vol. 280; no. 27; pp. 25313 - 25322
• Main Authors: Macauley, Matthew S, Whitworth, Garrett E, Debowski, Aleksandra W, Chin, Danielle, Vocadlo, David J
• Format: Journal Article
• Language: English
• Published: United States 08.07.2005
• Subjects: Acetylglucosamine - analogs & derivatives; Acetylglucosamine - pharmacology; Acetylglucosaminidase - antagonists & inhibitors; Acetylglucosaminidase - metabolism; Acetyltransferases - antagonists & inhibitors; Acetyltransferases - metabolism; Animals; beta-N-Acetylhexosaminidases - antagonists & inhibitors; beta-N-Acetylhexosaminidases - metabolism; Catalysis; Chlorocebus aethiops; COS Cells; Enzyme Activation - drug effects; Enzyme Activation - physiology; Glycoconjugates - metabolism; Histone Acetyltransferases; Humans; Lysosomes - enzymology; Multienzyme Complexes - antagonists & inhibitors; Multienzyme Complexes - metabolism; Substrate Specificity; Thiazoles - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M413819200

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.