Format

Send to

Choose Destination
See comment in PubMed Commons below
Biochemistry. 1993 Jul 6;32(26):6624-31.

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.

Author information

  • 1Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.

Abstract

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.

PMID:
8329389
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Loading ...
    Write to the Help Desk