α-Secondary Isotope Effects in Reactions of Exo-α-Glucanases

Exo-α-glucanases (β-amylase, glucoamylase, glucodextranase) that catalyze the hydrolysis of specific a-glucosidic substrates with inversion of configuration have usually been assumed to act by a base assisted nucleophilic displacement mechanism. On the other hand, the possibility of exo-carbonium ion mediation of such reaction has been recognized, but no supporting experimental evidence for this type of mechanism appears to have been reported. In order to examine this possibility, we have studied the α-secondary hydrogen isotope effects on the hydrolysis of α-glucosyl fluoride catalyzed by glucoamylases of several origins, and by a glucodextranase. α-Secondary deuterium isotope effects were 1.11-1.26 on these reactions. α-Secondary tritium isotope effects ranging from 1.17 to 1.26 were also measured for the hydrolysis of α-glucosyl fluoride catalyzed by these exo-α-glucanases. These results indicate that the reactions of hydrolysis of the C-F glycosylic bond of glycosyl fluoride by glucoamylase and glucodextranase proceed via an intermediate with oxo-carbonium ion character.