3 HISTIDINE-RESIDUES IN THE ACTIVE-CENTER OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM ALKALOPHILIC BACILLUS SP-1011 - EFFECTS OF THE REPLACEMENT ON PH-DEPENDENCE AND TRANSITION-STATE STABILIZATION

Cyclodextrin glucanotransferase (CGTase) catalyzes the formation of cy clodextrins from amylose through an intramolecular transglycosylation reaction. On the basis of the three-dimensional structures of CGTases three histidine residues, which are conserved between CGTases and alph a-amylases, are located at the active center and are proposed to const itute the substrate binding sites. The three histidine residues (His-1 40, His-233, and His-327) of CGTase from alkalophilic Bacillus sp. 101 1 were individually replaced by site-directed mutagenesis to probe the ir roles in catalysis. Asparagine-replaced CGTases (H140N-, H233N-, an d H327N-CGTase) retained cyclization activity but had altered producti on ratios of alpha-, beta-, and gamma-cyclodextrin. Replacement of his tidine by asparagine residues strongly affected the k(cat) for beta-cy clodextrin-forming, coupling, and hydrolyzing activities, whereas it b arely affected the K(m) values. The activation energies for alpha-cycl odextrin hydrolysis were increased more than 12 kJ/mol by the replacem ent. Furthermore, the K(i) values of acarbose, which is thought to be a transition-state analog of glycosidase catalysis, were 2-3 orders of magnitude larger in asparagine-replaced CGTases than that in wild-typ e CGTase. Therefore, the three histidine residues participate in the s tabilization of the transition state, whereas they participate little in ground-state substrate binding. H327N-CGTase had decreased activity over an alkaline pH range, indicating that His-327 is important for c atalysis over an alkaline pH range.