4 AROMATIC RESIDUES IN THE ACTIVE-CENTER OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM ALKALOPHILIC BACILLUS-SP-1011 - EFFECTS OF REPLACEMENTS ONSUBSTRATE-BINDING AND CYCLIZATION CHARACTERISTICS

Three-dimensional structures of cyclodextrin glucanotransferases (CGTa ses) have revealed that four aromatic residues, which are highly conse rved among CGTases but not found in alpha-amylases, are located in the active center. To analyze the roles of these aromatic residues, Phe-1 83, Tyr-195, Phe-259, and Phe-283 of Bacillus sp. 1011 CGTase were rep laced by site-directed mutagenesis, and the effects of this procedure were examined. Y195L-CGTase, in which Tyr-195 was replaced by a leucin e residue, underwent a drastic change in its cyclization characteristi cs: it produced considerably more gamma-cyclodextrin than the wild-typ e enzyme and virtually no alpha-cyclodextrin. Y195L-CGTase had increas ed K-m values for cyclodextrins, whereas the values for a linear malto oligosaccharide donor were insignificantly changed. Taken together wit h the structural information of CGTase crystals soaked with substrates , we propose that Tyr-195 plays an important role in the spiral bindin g of substrate. Replacing either Phe-183 or Phe-259 with leucine induc ed increased K-m values for accepters. Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal. Th ese results indicated that Phe-183 and Phe-259 are cooperatively invol ved in acceptor binding, and that they play a critical role in cycliza tion when the nonreducing end of amylose binds to the active center of CGTase. Replacing Phe-283 with a leucine residue induced a decrease i n k(cat) and in affinity for acarbose, suggesting that Phe-283 is invo lved in transition-state stabilization.