Abstract
Aspergillus niger glucoamylase (GA) consists of a catalytic domain and a starch-binding domain (SBD). SBD has two starch-binding sites, Sites 1 and 2. SBD can be isolated as a fragment protein. This calorimetric study aimed to examine the possibility of interactions between the two domains in a GA molecule and between the two binding sites of SBD [Biochemistry 38, 6300 (1999) and Eur. J. Biochem. 225, 133 (1994)] upon binding of a substrate analog, β-cyclodextrin (β-CD). The thermodynamic parameters of the binding of β-CD to the SBD fragment were virtually the same as those to SBD in the GA molecule. The values of the dissociation constants were 12–14 μM for Site 1 and 2 μM for Site 2, irrespective of the SBD fragment and SBD in the GA molecule. This result suggested that there was no inter-domain interaction between SBD and the catalytic domain for the binding of β-CD. In addition, amino acid mutation at one binding site did not affect the thermodynamic parameters at the other site. It was found that there is no interaction between the two binding sites in SBD and β-CD binds to Sites 1 and 2 independently.
https://ror.org/01529vy56 
