Wu J W, Wang Z X, Zhou J M
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Biochim Biophys Acta. 1997 Nov 14;1343(1):107-16. doi: 10.1016/s0167-4838(97)00089-7.
The unfolding behavior of dihydrofolate reductase from Chinese hamster in solutions of guanidine hydrochloride (GdnHCl) was studied. The GdnHCl-induced unfolding of the dihydrofolate reductase monitored by intrinsic fluorescence shows a biphasic transition, while the change in the enzyme activity is a single exponential process. The rate constant of inactivation is consistent with that of the fast conformational change. Therefore, the kinetic intermediate of protein unfolding should be a partially folded and inactive form. On the basis of the kinetic equation of substrate reaction in the presence of GdnHCl, all microscopic kinetic constants for the free enzyme and enzyme-substrate complexes have been determined. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation.
研究了中国仓鼠二氢叶酸还原酶在盐酸胍（GdnHCl）溶液中的展开行为。通过内源荧光监测的GdnHCl诱导的二氢叶酸还原酶展开呈现双相转变，而酶活性的变化是单指数过程。失活速率常数与快速构象变化的速率常数一致。因此，蛋白质展开的动力学中间体应该是部分折叠且无活性的形式。基于存在GdnHCl时底物反应的动力学方程，确定了游离酶和酶 - 底物复合物的所有微观动力学常数。两种底物，NADPH和7,8 - 二氢叶酸，都能保护二氢叶酸还原酶不被失活。
