Jiang Wei, Han Yingchun, Pan Qunhui, Shen Tao, Liu Changlin
Department of Chemistry, Huazhong University of Science and Technology, Wuhan 430074, China.
J Inorg Biochem. 2007 Apr;101(4):667-77. doi: 10.1016/j.jinorgbio.2006.12.011. Epub 2007 Jan 3.
It is well known that the wild type Cu,Zn superoxide dismutase (holo SOD) catalyzes the conversion of superoxide anion to peroxide hydrogen and dioxygen. However, a new function of holo SOD, i.e., nucleolytic activity has been found [W. Jiang, T. Shen, Y. Han, Q. Pan, C. Liu, J. Biol. Inorg. Chem. 11 (2006) 835-848], which is linked to the incorporation of exogenous divalent metals into the enzyme-DNA complex. In this study, the roles of exogenous divalent metals in the nucleolytic activity were explored in detail by a series of biochemical experiments. Based on a non-equivalent multi-site binding model, affinity of a divalent metal for the enzyme-DNA complex was determined by absorption titration, indicating that the complex can provide at least a high and a low affinity site for the metal ion. These mean that the holo SOD may use a "two exogenous metal ion pathway" as a mechanism in which both metal ions are directly involved in the catalytic process of DNA cleavage. In addition, the pH versus DNA cleavage rate profiles can be fitted to two ionizing-group models, indicating the presence of a general acid and a general base in catalysis. A model that requires histidine residues, metal-bound water molecules and two hydrated metal ions to operate in concert could be used to interpret the catalysis of DNA hydrolysis, supported by the dependences of loss of the nucleolytic activity on time and on the concentration of the specific chemical modifier to the histidine residues on the enzyme.
众所周知，野生型铜锌超氧化物歧化酶（全酶SOD）催化超氧阴离子转化为过氧化氢和氧气。然而，已发现全酶SOD的一种新功能，即核酸裂解活性[W. Jiang, T. Shen, Y. Han, Q. Pan, C. Liu, J. Biol. Inorg. Chem. 11 (2006) 835 - 848]，这与外源二价金属掺入酶 - DNA复合物有关。在本研究中，通过一系列生化实验详细探究了外源二价金属在核酸裂解活性中的作用。基于非等效多位点结合模型，通过吸收滴定法测定了二价金属对酶 - DNA复合物的亲和力，表明该复合物可为金属离子提供至少一个高亲和力位点和一个低亲和力位点。这意味着全酶SOD可能使用“双外源金属离子途径”作为一种机制，其中两种金属离子都直接参与DNA裂解的催化过程。此外，pH与DNA裂解速率曲线可拟合为两个电离基团模型，表明催化过程中存在一个广义酸和一个广义碱。一个需要组氨酸残基、金属结合水分子和两个水合金属离子协同作用的模型可用于解释DNA水解的催化作用，酶上组氨酸残基的核酸裂解活性丧失对时间和特定化学修饰剂浓度的依赖性支持了这一模型。
