| Abstract
| - Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficientlycatalyze transglycosylation reactions. The mechanism of the glycosynthase reaction is probed with theE134A mutant of Bacilluslicheniformis 1,3-1,4-β-glucanase. This endo-glycosynthase is regiospecificfor formation of a β-1,4-glycosidic bond with α-glycosyl fluoride donors (laminaribiosyl as the minimaldonor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (arylmonosaccharides or oligosaccharides). The pH dependence of the glycosynthase activity reflects generalbase catalysis with a kinetic pKa of 5.2 ± 0.1. Kinetics of enzyme inactivation by a water-solublecarbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pKa of5.3 ± 0.2. The general base is Glu138 (the residue acting as the general acid−base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A. It is devoid of glycosynthaseactivity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a β-glycosylazide product. The pKa of Glu138 (kinetic pKa on kcat/KM and pKa of EDC inactivation) for the E134Aglycosynthase has dropped 1.8 pH units compared to the pKa values of the wild type, enabling the sameresidue to act as a general base in the glycosynthase enzyme. Kinetic parameters of the E134Aglycosynthase-catalyzed condensation between Glcβ4Glcβ3GlcαF (2) as a donor and Glcβ4Glcβ-pNP(15) as an acceptor are as follows: kcat = 1.7 s-1, KM(acceptor) = 11 mM, and KM(donor) < 0.3 mM.Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions forpreparative use of the glycosynthase reaction in oligosaccharide synthesis. Yields are 70−90% with arylmonosaccharide and cellobioside acceptors, but 25−55% with laminaribiosides, the lower yields (andlower initial rates) due to competitive inhibition of the β-1,3-linked disaccharide acceptor for the donorsubsites of the enzyme.
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