Subsite Structure and Mechanism of β-Glucosidase from Aspergillus niger
スポンサーリンク
概要
- 論文の詳細を見る
The β-glucosidase (EC 3.2.1.21, βGA) preparation purified from Aspergillus niger (Novozym 188DCN0003) was confirmed to be a monomer protein of Mw 137, 000. With this preparation, binding of glucono-l:5-lactone (GLN) to βGA was studied using fluorescence-spectrophotometric and stopped flow kinetic experiments on the basis of Trp-residue (s), and an inhibition kinetic experiment for the cellobiose substrate. The dissociation constant (K<SUB>d</SUB>) and inhibitor constant (K<SUB>i</SUB>) (competitive inhibition) for the GLN-βGA complex were evaluated to be 10μM and 15μM, respectively. The subsite structure of βGA was analyzed with the steady-state kinetic method. It was confirmed that the active site was composed of six subsites, affinity at subsite 2 (A<SUB>2</SUB>) was the largest and subsites 4-6 had negative affinity. We tried to interpret the binding of GLN on the basis of the subsite structure, where subsite 1 carried an indispensable role in the productive binding mode. Determining the kinetic parameters for several β-glucosides as substrates, it was shown that the molar activity (k<SUB>0</SUB>) and Michaelis constant (K<SUB>m</SUB>) were very characteristic of the substituent species in aglycone phenyl-residue. Furthermore, the value of k<SUB>0</SUB>/K<SUB>m</SUB>, which reflects the productive-binding mode, strongly depends on substituent constant n (degree of hydrophobicity), suggesting that subsite 2, in which the aglycone residue is bound, is intimately related to the "hydrophobic-driven" mechanism for ES-complex formation. Further, the effect of acetonitrile (0-32.3%) on the hydrolytic reaction was examined for the cellobiose substrate. The results support the hydrophobic-driven mechanism.
- 日本応用糖質科学会の論文
日本応用糖質科学会 | 論文
- 古細菌の産生する新規な糖質関連酵素およびそれらを用いたトレハロースの生産
- PCR法による米 (Oryza sativa L.) の品種判別およびその米加工品への応用
- モチ米のアミロペクチン鎖長分布と米粒の物理特性との関係
- ジャポニカ米とインデカ米の澱粉とプロテインボディの結合脂質について
- 促通拡散型糖輸送担体分子の構造機能相関