Calorimetric Study on the Binding of p-Cyclodextrin to the Starch-binding Domain of Aspergillus niger Glucoamylase

摘要

黒コウジ力ビのダルコアミラーゼは触媒ドメインとデンプン結合ドメイン（SBD)から構成される。SBDには2つ の基質結合部位がある。本論文では，リガンド結合に関し て2つのドメィン間および2か所の結合部位間に相互作用 [Biochemistry 38, 6300 (1999), Eur. J. Biochem. 225，133 (1994)]があるかどうかを，基質アナログであるβ-シクロデ キストリン（β-CD)との結合の熱力学量を指標に検討を行 つた。全長酵素およびSBDフラグメント（SBDF)におい て，P-CDとの結合の熱力学量に差は見られなかった。また SBDFについて，1つの結合部位の疎水性ァミノ酸残基を置 換しても，他方の部位の結合の熱力学量への影響はなかつ た。これらの結果から，リガンド結合に関して2つのドメ ィン間および2つの結合部位間に相互作用はないと考えら れた。%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, p-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 1214 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 p-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 p-CD binds to Sites 1 and 2 independently.