SUGAR BINDING IN CRYSTALLINE CYCLODEXTRIN GLUCANOTRANSFERASE. K. Harata¹, N. Ishii¹, K. Haga², M. Aoyagi², K. Yamane², ¹National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan, and ²Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Cyclodextrin glucanotransferase (CGTase) is an enzyme which produces cyclodextrins by the degradation of starch. The enzyme from alkalophilic Bacillus sp.1011 is stable in markedly wide pH range. To elucidate the mechanism of enzyme action, the crystal structure of the acarbose complex has been investigated by the X-ray method. The acarbose molecule is bound in the active center and has short contacts with Asp229, Glu257, and Asp 328 which have been considered as catalytic residues. Asp229 and Glu257 are located at the secondary hydroxyl side of acarbose in the distance of hydrogen-bonding contact with a glycosidic oxygen atom. On the other hand, Asp328 is hydrogen-bonded to O5 of the adjacent pyranose ring. The result is consistent with the proposed model of the catalytic reaction. The crystal contains two independent CGTase molecules. In both molecules, acarbose is bound in the same manner, but differences are observed in contacts with individual amino acid residues.

Crystal of the complex was obtained by the co-crystallization in the presence of 1mM acarbose in the same condition as used in the crystallization of isomorphous native crystal. The structure was refined to the current R-value of 0.17 for the 2.2Å resolution data. The structural study of acarbose complexes of CGTase mutants are in progress.