Shinya Shoko, Ghinet Mariana G, Brzezinski Ryszard, Furuita Kyoko, Kojima Chojiro, Shah Sneha, Kovrigin Evgenii L, Fukamizo Tamo
Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara, 631-8505, Japan.
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
J Biomol NMR. 2017 Apr;67(4):309-319. doi: 10.1007/s10858-017-0109-6. Epub 2017 Apr 9.
Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.
通过核磁共振氢碳化学位移相关谱（HSQC）滴定实验中的谱线形状分析，研究了壳聚糖与壳聚糖酶的相互作用。我们确定，底物壳聚糖六聚体通过三态诱导契合机制与酶结合，首先快速形成遭遇复合物，随后结合态缓慢异构化为最终构象。该机制两个连续步骤中的化学位移扰动图谱突出显示了底物结合亚位点和铰链区参与了结合反应。三态模型的平衡参数与等温滴定量热法（ITC）测定的整体热力学解离常数一致。本研究提供了糖苷水解酶中诱导契合机制的首个动力学证据。
