title: Detailed comparative analysis of the catalytic mechanisms of beta-N-acetylglucosaminidases from families 3 and 20 of glycoside hydrolases.
abstract: Beta-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. Alpha-secondary deuterium kinetic isotope effects of kH/kD = 1.07 and 1.10 for Streptomyces plicatus beta-hexosaminidase (SpHex) and Vibrio furnisii beta-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 beta(lg) (-0.29) on kcat/Km, compared with a beta(lg) 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.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry; D007700:Kinetics; D010100:Oxygen / Q000737:chemistry; D011522:Protons; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology
chemical_list: D011522:Protons; D003903:Deuterium; D000118:Acetylglucosaminidase; D010100:Oxygen