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 broa d substrate specificity and has been implicated in the transformation of xe nobiotic glycosides, CBG also catalyzes a variety of transglycosylation rea ctions, which have been been shown with other glycosylhydrolases to functio n in synthetic and genetic regulatory pathways. We investigated the catalyt ic mechanism, substrate specificity, and transglycosylation acceptor specif icity of guinea pig liver CBG by several methods. These studies indicate th at CBG employs a two-step catalytic mechanism with the formation of a coval ent enzyme-sugar intermediate and that CBG mill transfer sugar residues to primary hydroxyls and equatorial but not axial C-4 hydroxyls of aldopyranos yl sugars, Kinetic studies revealed that correction for transglycosylation reactions is necessary to derive correct kinetic parameters for CBG. Furthe r analyses revealed that for aldopyranosyl substrates, the activation energ y barrier is affected most by the presence of a C-6 carbon and by the confi guration of the C-2 hydroxyl, whereas the binding energy is affected modest ly by the configuration and substituents at C-2, C-4, and C-5. These data i ndicate that the transglycosylation activity of CBG derives from the format ion of a covalently linked enzyme-sugar intermediate and that the specifici ty of CBG; for transglycosylation reactions is different from its specifici ty for hydrolysis reactions.