Catalytic mechanism and specificity for hydrolysis and transglycosylation reactions of cytosolic beta-glucosidase from guinea pig liver.

Cytosolic beta-glucosidase (CBG) from mammalian liver is known for its broad substrate specificity and has been implicated in the transformation of xenobiotic glycosides. CBG also catalyzes a variety of transglycosylation reactions, which have been been shown with other glycosylhydrolases to function in synthetic and genetic regulatory pathways. We investigated the catalytic mechanism, substrate specificity, and transglycosylation acceptor specificity of guinea pig liver CBG by several methods. These studies indicate that CBG employs a two-step catalytic mechanism with the formation of a covalent enzyme-sugar intermediate and that CBG will transfer sugar residues to primary hydroxyls and equatorial but not axial C-4 hydroxyls of aldopyranosyl sugars. Kinetic studies revealed that correction for transglycosylation reactions is necessary to derive correct kinetic parameters for CBG. Further analyses revealed that for aldopyranosyl substrates, the activation energy barrier is affected most by the presence of a C-6 carbon and by the configuration of the C-2 hydroxyl, whereas the binding energy is affected modestly by the configuration and substituents at C-2, C-4, and C-5. These data indicate that the transglycosylation activity of CBG derives from the formation of a covalently linked enzyme-sugar intermediate and that the specificity of CBG for transglycosylation reactions is different from its specificity for hydrolysis reactions.