Three 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 cyclodextrins 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 alpha-amylases, are located at the active center and are proposed to constitute the substrate binding sites. The three histidine residues (His-140, His-233, and His-327) of CGTase from alkalophilic Bacillus sp. 1011 were individually replaced by site-directed mutagenesis to probe their roles in catalysis. Asparagine-replaced CGTases (H140N-, H233N-, and H327N-CGTase) retained cyclization activity but had altered production ratios of alpha-, beta-, and gamma-cyclodextrin. Replacement of histidine by asparagine residues strongly affected the kcat for beta-cyclodextrin-forming, coupling, and hydrolyzing activities, whereas it barely affected the Km values. The activation energies for alpha-cyclodextrin hydrolysis were increased more than 12 kJ/mol by the replacement. Furthermore, the Ki 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-type CGTase. Therefore, the three histidine residues participate in the stabilization 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 catalysis over an alkaline pH range.