Bacillus 1,3-1,4-β-glucanases hydrolyze 1,3-1,4-β-gluco-oligosaccharides with a retaining mechanism. The binding-site cleft of these endoglycosidases is composed of six subsites (-4 to + 2) of which subsite - 3 makes the largest contribution to transition state stabilization. The specificity of this subsite is here analyzed for both glycosidase and glycosynthase activities in the wild-type and the nucleophile-less E134A mutant Bacillus licheniformis enzymes. A D-galactosyl residue on the nonreducing end of a trisaccharide substrate is accepted by the enzyme and binds at subsite - 3 in the productive enzyme-substrate complex. The wild-type enzyme catalyzes the hydrolysis of the substrate Glcβ4Glcβ3GlcβMU (Glc=glucosyl, MU = 4-methylumbelliferyl) with a kcat/KM, value only 1.3-fold higher than for the Galβ4Glcβ3GlcβMU (Gal=galactosyl) substrate. The corresponding α-fluorides act as good donors for the glycosynthase condensation reaction with mono- and disaccharide acceptors catalyzed by the E134A mutant. Whereas self-condensation and elongation products are also obtained as minor compounds with the Glcβ4Glcβ3GlcαF donor, nearly quantitative yields of single condensation products are obtained with the Galβ4Glcβ3GlcαF donor, in which the axial configuration of the 4-OH group on the nonreducing end prevents self-condensation and elongation reactions.
|Nombre de pàgines||8|
|Estat de la publicació||Publicada - 2002|