Detailed Comparative Analysis of the Catalytic Mechanisms of β-N-Acetylglucosaminidases from Families 3 and 20 of Glycoside Hydrolases
journal contributionposted on 27.09.2005, 00:00 by David J. Vocadlo, Stephen G. Withers
β-N-Acetylglucosaminidases are commonly occurring enzymes involved in the degradation of polysaccharides and glycoconjugates containing N-acetylglucosamine residues. Such enzymes have been classified into glycoside hydrolase families 3 and 20 and are believed to follow distinct chemical mechanisms. Family 3 enzymes are thought to follow a standard retaining mechanism involving a covalent glycosyl enzyme intermediate while family 20 enzymes carry out a substrate-assisted mechanism involving the transient formation of an enzyme-sequestered oxazoline or oxazolinium ion intermediate. Detailed mechanistic analysis of representatives of these two families provides support for these mechanisms as well as detailed insights into transition state structure. α-Secondary deuterium kinetic isotope effects of kH/kD = 1.07 and 1.10 for Streptomyces plicatus β-hexosaminidase (SpHex) and Vibrio furnisii β-N-acetylglucosaminidase (ExoII) respectively indicate transition states with oxocarbenium ion character in each case. Brønsted plots for hydrolysis of a series of aryl hexosaminides are quite different in the two cases. For SpHex a large degree of proton donation is suggested by the relatively low value of βlg (−0.29) on kcat/Km, compared with a βlg of −0.79 for ExoII. Most significantly the Taft plots derived from kinetic parameters for a series of p-nitrophenyl N-acyl glucosaminides bearing differing levels of fluorine substitution in the N-acyl group are completely different. A very strong dependence (slope = −1.29) is seen for SpHex, indicating direct nucleophilic participation by the acetamide, while essentially no dependence (0.07) is seen for ExoII, suggesting that the acetamide plays purely a binding role. Taken together these data provide unprecedented insight into enzymatic glycosyl transfer mechanisms wherein the structures of both the nucleophile and the leaving group are systematically varied.
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insighttransition state structurefamily 3 enzymeschemical mechanismsproton donationfamily 20 enzymesnucleophilic participationTaft plotsCatalytic MechanismsseriesAcetylglucosaminidaseoxazolinium ionComparative Analysisglycosyl transfer mechanismsacetamideBr ønsted plotsSpHexdependenceacetylglucosamine residuescovalent glycosyl enzymeβ lgtransition statesglycoside hydrolase families 3nitrophenyl Noxocarbenium ion characteracyl glucosaminidesacyl groupisotope effectsbinding roleExoIISuch enzymesaryl hexosaminidesfluorine substitutionFamilies 3