A critical evaluation of metal-promoted Klenow 3′-5′ exonuclease activity:: calorimetric and kinetic analyses support a one-metal-ion mechanism
A critical evaluation of metal-promoted Klenow 3′-5′ exonuclease activity:: calorimetric and kinetic analyses support a one-metal-ion mechanism
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DOI：
10.1007/s007750050234
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发表时间：
1998-06-01
影响因子：
3
通讯作者：
Cowan, JA
中科院分区：
文献类型：
--
作者：
Black, CB;Cowan, JA
金属介导的磷酸酯水解是核酸生物化学中一种常见的催化途径。在对镁依赖性核酸酶活化的机理讨论中，主要援引了两个不同的模型：即。一个和两个金属离子途径。大肠杆菌DNA聚合酶I的Klenow片段的3‘-5’外切酶结构域是双金属离子机制的范例：然而，该反应模型主要基于在掺杂实验中使用高浓度过渡金属类似物和高浓度背景硫酸铵的结构和动力学实验。这促使我们通过溶液动力学和等温滴定量热法，利用天然Mg2+辅因子和盐条件，重新评估了Klenow片段的3‘-5’外切酶机制的金属辅因子化学计量学。溶液量热和动力学实验都强烈表明，只有一种金属离子与外切酶活性位点结合。与Mn2+的比较研究也表明需要一种金属离子来影响3‘-5’外切酶活性。
Metal-mediated hydrolysis of phosphate esters is a common catalytic pathway in nucleic acid biochemistry. Two distinct models are principally invoked in mechanistic discussions of these reactions for magnesium-dependent nuclease activation: namely. the one-versus two-metal-ion pathways. The 3'-5' exonuclease domain of the Klenow fragment of Escherichia coli DNA polymerase I is a paradigm for the two-metal-ion mechanism: however, this reaction model is principally based on structural and kinetics experiments employing high concentrations of transition metal analogues and high concentrations of background ammonium sulfate during doping experiments. This prompted us to reevaluate the metal cofactor stoichiometry of the 3'-5' exonuclease mechanism for the Klenow fragment by solution kinetics and isothermal titration calorimetry using the natural Mg2+ cofactor and salt conditions. Both solution calorimetric and kinetics experiments strongly indicate binding of only one metal ion to the exonuclease active site. Comparative studies with Mn2+ also indicate a requirement for one metal ion to effect 3'-5' exonuclease activity.