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.
Nakamura
A
A
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Haga
K
K
Yamane
K
K
D020909:Acarbose; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014312:Trisaccharides; D006639:Histidine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D020909:Acarbose