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Abstract

The exoglycanase from Cellulomonas fimi catalyses the hydrolysis of cello oligosaccharides to cellobiose as well as the hydrolysis of xylan and aryl β-glycosides (Gilkes et al (1984) J. Biol. Chem. 259, 10455). Its mechanism of action is thought to involve a double displacement reaction which is investigated here through detailed kinetic studies of the native enzyme and point mutants with a range of aryl β-glycosides, and through inactivation studies with 2-deoxy- and 2-deoxyfluoro-glycoside mechanism-based inactivators and the affinity label, N-bromoacetyl cellobiosylamine. A pH study of the native enzyme revealed ionisations of PKa = 4.1 and 7.7 in the free enzyme, likely corresponding to the catalytic nucleophile and the acid-base catalyst, respectively. The large secondary deuterium kinetic isotope effects measured on both steps for the glucosides and on the deglycosylation step for the cellobiosides reveal significant oxocarbonium ion character at the corresponding transition states, thus suggesting substantial C-O bond cleavage and little nucleophilic preassociation. By contrast, the relatively small secondary deuterium kinetic isotope effect and the small Broensted constant measured on the glycosylation step for the cellobiosides suggest that the cellobiosylation transition state is less highly charged than the glucosylation transition state. These studies suggest that the primary function of the distal glucosyl moiety of the cellobiosides is to increase the rate of glycosylation, likely through improved acid catalysis and greater nucleophile preassociation, without affecting its rate of deglycosylation. The greater rates of hydrolysis of the xylo-sugars, relative to those for the gluco-sugars, indicate that the substrate preference of C. fimi exoglycanase increases in the order glucosides