The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.
Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow 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 mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.
van der Veen
B A
BA
1Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan, NN Haren, The Netherlands.
van Alebeek
G J
GJ
Uitdehaag
J C
JC
Dijkstra
B W
BW
Dijkhuizen
L
L
D001407:Bacillus / Q000201:enzymology*; D020134:Catalytic Domain; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D013379:Substrate Specificity
D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase