Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase

Cyclodextrin-glycosyltransferases (CGTases) (EC 2.4.1.19) preferably cataly ze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous alpha -amylases catalyze hydrolysis reactions using water a s acceptor. This difference in reaction specificity is most likely caused b y the acceptor binding site. To investigate this in detail we altered the a cceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus ci rculans strain 251 CGTase using site-directed mutagenesis. Lys-232 is of ge neral importance for catalysis, which appears to result mainly from stabili zation of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state s tabilization. Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor. Phe-183 and Phe-259 play important and distinct roles in the transglycosylation rea ctions catalyzed by CGTase. Mutation of Phe-183 affects especially the cycl ization and coupling reactions, whereas Phe-259 is most important for the c yclization and disproportionation reactions. Moreover, the hydrophobisity o f Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme. Hydrol ysis can be enhanced by making these residues more polar, which concomitant ly results in a lower transglycosylation activity. A double mutant was cons tructed that yielded an enzyme preferring hydrolysis over cyclization (15:1 ), whereas the wild type favors cyclization over hydrolysis (90:1).