Fan Y X, Ju M, Zhou J M, Tsou C L
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Biochim Biophys Acta. 1995 Sep 27;1252(1):151-7. doi: 10.1016/0167-4838(95)00125-e.
The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3-fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed.
将鸡肝二氢叶酸还原酶在较宽尿素浓度范围内变性过程中的激活与失活，与内在荧光的变化进行了比较。在2 M尿素中，该酶被激活3.6倍，并且以激活形式稳定长达12小时。在4 M尿素中，酶活性最初增加约5倍，但激活的酶迅速失活至远低于天然酶的水平。在DHF或NADPH存在下，激活的酶得以稳定。测定并比较了该酶在不同尿素浓度下对底物的解离常数（Kd）和米氏常数（Km）。在3 M尿素存在下，DHF和NADPH的Kd值分别增加4倍和10倍，而相应的Km值分别增加25倍和3倍。最大反应速度（Vmax）的大幅增加是激活的主要原因。尿素中的失活和去折叠都是双相过程。对于快速相，在4 M尿素中，失活的速率常数比去折叠的速率常数大10倍。还研究了硫酸铵对该酶激活和去折叠的影响。结果表明，该酶的活性位点更容易受到变性剂的干扰；并且激活的酶在活性位点似乎具有更开放和灵活的构象，这有利于酶催化能力的充分发挥。提出了一个随着尿素浓度增加，二氢叶酸还原酶（DHFR）在去折叠过程中依次激活和失活的示意图。
