The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylatio n reactions via a double displacement mechanism involving a covalent enzyme -intermediate complex (substituted-enzyme intermediate). Characterization o f the three transglycosylation reactions, however, revealed that they diffe r in their kinetic mechanisms. Disproportionation (cleavage of an alpha-gly cosidic bond of a linear malto-oligosaccharide: and transfer of one pad to an acceptor substrate) proceeds according to a ping-pong mechanism. Cycliza tion (cleavage of an alpha-glycosidic bond in amylose or starch and subsequ ent formation of a cyclodextrin) is a single-substrate reaction with an aff inity for the high molecular mass substrate used, which was too high to all ow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however , have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mec hanisms observed for the various reactions, which can be related to differe nces in substrate binding, it should be possible to mutagenize CGTase in su ch. a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclo dextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization a nd coupling reactions was found to determine the reaction rate. In the cycl ization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of th e enzyme. These new insights will allow rational design of CGTase mutant en zymes synthesizing cyclodextrins of specific sizes.