Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.
O-GlcNAcase is a family 84 beta-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of beta-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 Asp(174) as the residue that polarizes the 2-acetamido group for attack on the anomeric center and Asp(175) 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.
Cetinbaş
Naniye
N
Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada, V5A 1S6.
Macauley
Matthew S
MS
Stubbs
Keith A
KA
Drapala
Robert
R
Vocadlo
David J
DJ
D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000136:Acid-Base Equilibrium; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013865:Thioglycosides / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases
D001386:Azides; D009097:Multienzyme Complexes; D013865:Thioglycosides; D001224:Aspartic Acid; D051548:Histone Acetyltransferases; C110628:hexosaminidase C; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases