|Title||Detailed comparative analysis of the catalytic mechanisms of beta-N-acetylglucosaminidases from families 3 and 20 of glycoside hydrolases|
|Publication Type||Journal Article|
|Year of Publication||2005|
|Authors||Vocadlo, DJ, Withers, SG|
|Date Published||SEP 27|
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 k(H)/k(D) = 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. Bronsted 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 P-lg (-0.29) on k(cat)/K-m, 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.