O-GlcNAcase Uses Substrate-assisted Catalysis

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 ␤-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. Humans have three genes encoding enzymes that cleave terminal N-acetylglucosamine residues from glycoconjugates. The first of these, O-GlcNAcase, 1 is a member of family 84 of glycoside hydrolases that includes enzymes from organisms as diverse as prokaryotic pathogens and humans (1, 2). 2 The substrates of O-GlcNAcase are post-translationally modified glycoproteins bearing the monosaccharide 2-acetamido-2-deoxy-␤-D-glucopyranoside linked to serine or threonine residues (O-GlcNAc) (3-5). This post-translational modification is abundant in mammalian cells (3) and is found on many cellular proteins having a wide range of vital cellular functions, including for example, transcription (6 -9), proteasomal degradation (10), and cellular signaling (11), and is also found on many structural proteins (12)(13)(14). Consistent with the abundance of O-GlcNAc on intracellular proteins, it appears to have roles in the etiology of several disease states, including type II diabetes (11, 15), Alzheimer (13,16,17), and cancer (18). Although O-GlcNAcase was likely isolated earlier on (19,20), an understanding of its biochemical role in acting to cleave O-GlcNAc off from modified serine and threonine residues of proteins waited some 20 years (21). More recently OGlcNAcase has been cloned (23), partially characterized (24), and suggested to have additional activity as a histone acetyltransferase (22). Little, however, is known about the catalytic mechanism of this enzyme.HEXA and HEXB are the two other genes that encode enzymes catalyzing the hydrolytic cleavage of terminal N-acetylglucosamine residues from glycoconjugates. The gene products of HEXA and HEXB predominantly yield two dimeric isozymes. The homodimeric isozyme, hexosaminidase B (␤␤), is composed of two ␤-subunits, and the heterodimeric isozyme, hexosaminidase A (␣␤), is composed of an ␣-and a ␤-subunit. Both of these enzymes are normally localized within the lysosome. The two subunits bear a high level of sequence identity, and both are members of family 20 of glycoside hydrolases. The dysfunction of...

Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases

“…The value we observe most closely resembles that seen for GH84 human O-GlcNAcase ( ranging from Ϫ0.42 to Ϫ1.6 for GH84 enzymes) (15,19) but is significantly less that those observed for GH20 enzymes (* ranging from Ϫ1.0 to Ϫ1.29) (15, 37). The good correlation we observe here, coupled with the similarity of value to values observed for enzymes from GH84, provides strong kinetic evidence that these enzymes share in common a catalytic mechanism involving substrate assistance.…”

“…(Fig. 1D) is a potent inhibitor of glycoside hydrolases from GH20 (15,16) and GH84 (15) by virtue of its mimicry of the enzyme-catalyzed transition state (60) or the high energy oxazoline intermediate (Fig. 1B).…”

“…1B) are intermediates within the catalytic cycle of glycoside hydrolases from families 18, 20, 56, and 84. Enzymes from families 18, 20, 56, and 84 have previously been shown to use substrate-assisted catalysis through both detailed kinetic studies (15)(16)(17)(18)) and x-ray structural studies (19 -23). Therefore, the ability of GH85 endohexosaminidases to use sugar oxazoline donors strongly suggests that GH85 enzymes also use a substrate-assisted catalytic mechanism.…”

Targeted in Vivo O-GlcNAc Sensors Reveal Discrete Compartment-specific Dynamics during Signal Transduction

“…Although only a single gene exists for each enzyme, both are known to have several splice variants. O-GlcNAcase has two splice variants, a full-length version, whose localization is mainly cytosolic, and a catalytically less active nuclear-localized variant that is missing a putative histone acetyltransferase domain (16). For OGT, three variants have been identified, short-and full-length nuclear/ cytoplasmic localized versions and a mitochondrial localized enzyme (17).…”

A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group of β-N-acetylglucosaminidases

“…1) has been found to be a potent inhibitor of family 20 hexosaminidases 2, 3 and, more recently, family 84 O-GlcNAcases. 5 Its poor inhibitory potency toward family 3 is consistent with this family of enzymes. O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino-Nphenylcarbamate (PUGNAc) 2, 12 the tetrahydroimidazopyridines, with the most important being 8-acetamido-5,6,7,8-tetrahydro-6,7-dihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-acetic acid, gluco-nagstatin 3, 13 and the triazoles, all prepared by Vasella et al, have all been shown to potently inhibit family 3 b-N-acetylglucosaminidases, 14 family 20 human b-hexosaminidases 9, 13 and family 84 O-GlcNAcases.…”