Zhang Wen-he, Qin San-bo, Zhang Hong-jie, Zhu Zhi-yong
Institute of Biophysics, Academy Sinica, 15 Datun Road, Beijing 100101, China.
Protein Pept Lett. 2005 Jul;12(5):483-6. doi: 10.2174/0929866054395310.
The local fluorescence probes, 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and NADPH were employed to detect urea-induced conformation changes at each active site of dihydrofolate reductase (DHFR), respectively. The results indicate that local conformation change at DHF/TNS could be superimposed by the conformation change calculated from the enzyme activity change with a three-state model; while at NADPH site it is lagged in the first transition. This difference is further supported by the different relative changes of Michaelis constants at 0, 1 and 1.8 M urea for each substrate. Our results suggest that local conformation at DHF site is more flexible than that at NADPH site, and the urea-induced unfolding could be ascribed to a four-state transition.
使用局部荧光探针2-（对甲苯胺基）-6-萘磺酸（TNS）和NADPH分别检测二氢叶酸还原酶（DHFR）每个活性位点处尿素诱导的构象变化。结果表明，DHF/TNS处的局部构象变化可以通过用三态模型从酶活性变化计算出的构象变化叠加；而在NADPH位点，它在第一次转变中滞后。每个底物在0、1和1.8 M尿素下米氏常数的不同相对变化进一步支持了这种差异。我们的结果表明，DHF位点的局部构象比NADPH位点的更灵活，并且尿素诱导的去折叠可归因于四态转变。
