Reversible inactivation of alkaline phosphatase from Atlantic cod (Gadus morhua) in urea
Reversible inactivation of alkaline phosphatase from Atlantic cod (Gadus morhua) in urea
复制标题
DOI：
10.1016/j.bbapap.2005.12.015
复制
发表时间：
2006-02-01
影响因子：
3.2
通讯作者：
Guojónsdóttir, K
中科院分区：
文献类型：
--
作者：
Asgeirsson, B;Guojónsdóttir, K
来自大西洋鳕鱼（Gadus morhua）的碱性磷酸酶（AP）是一种含锌和镁的同型二聚体，需要低聚状态才能发挥活性。其动力学性质表明其具有冷适应性。在这里，研究尿素对结构稳定性的影响，以便将活性与金属含量、色氨酸残基周围的微环境和亚基界面事件联系起来。在尿素浓度最低时，首先检测到的性质变化是酶活性的增加。随后是失活，当尿素浓度达到2 M时，锌含量释放一半，内在色氨酸荧光和圆二色性椭圆度在2.5至8 M尿素范围内发生变化，表明二聚体解离，随后在6-8 M尿素发生一个主要单体展开转变，这是由ANS荧光和KI荧光猝灭所表明的。利用三态模型，用线性外推法估计了二聚体稳定性的吉布斯自由能为8.6 kcal/mol，单体展开的吉布斯自由能为11.6 kcal/mol，总自由能为31.8 kcal/mol。二聚体缔合的离子贡献很小。二聚体在相对高浓度的尿素环境下是稳定的，而活性位点附近则容易受到低浓度尿素的影响。因此，失活与二聚体解离并不一致，这表明活性位点是分子中最具活力的部分，与其酶活性的冷适应性最密切相关。(c) 2005 Elsevier b.v.版权所有
Alkaline phosphatase (AP) from Atlantic cod (Gadus morhua) is a zinc and magnesium containing homodimer that requires the oligomeric state for activity. Its kinetic properties are indicative of cold-adaptation. Here, the effect of urea on the structural stability was studied in order to correlate the activity with metal content, the microenvironment around tryptophan residues, and events at the subunit interface. At the lowest concentrations of urea, the first detected alteration in properties was an increase in the activity of the enzyme. This was followed by inactivation, and the release of half of the zinc content when the amount of urea reached levels of 2 M. Intrinsic tryptophan fluorescence and circular dichroism ellipticity changed in the range 2.5 to 8 M urea, signaling dimer dissociation, followed by one major monomer unfolding transition at 6-8 M urea as indicated by ANS fluorescence and KI fluorescence quenching. Gibbs free energy was estimated by the linear extrapolation method using a three-state model as 8.6 kcal/mol for dimer stability and 11.6 kcal/mol for monomer unfolding giving a total of 31.8 kcal/mol. Dimer association had a very small ionic contribution. Dimers were stable in relatively high concentration of urea, whereas the immediate vicinity around the active site was vulnerable to low concentrations of urea. Thus, inactivation did not coincide with dimer dissociation, suggesting that the active site is the most dynamic part of the molecule and closest related to cold-adaptation of its enzymatic activity. (c) 2005 Elsevier B.V All rights reserved.