Kinetic analysis of product release and metal ions in a metallonuclease.
Kinetic analysis of product release and metal ions in a metallonuclease.
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金属核酸酶中产物释放和金属离子的动力学分析。
DOI：
10.1016/j.abb.2009.01.001
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发表时间：
2009
影响因子：
3.9
通讯作者：
Dupureur,CynthiaM
中科院分区：
文献类型：
--
作者：
Xie,Fuqian;Dupureur,CynthiaM
大多数核酸酶依赖于二价阳离子作为辅因子来催化核酸磷酸二酯键的水解。在这里，平衡和动力学实验被用来测试最近提出的模型，关于产品释放的金属离子的依赖性和金属离子之间的cooperativity绑定在同二聚体PvuII核酸内切酶的活性位点的程度。平衡荧光各向异性的研究表明，在金属离子的存在下，产品的结合显着减弱。前稳态动力学表明，产品释放至少部分是速率限制的。稳态和预稳态数据最适合于其中金属在产品释放后保持与酶结合的模型。最后，合作和独立的金属离子结合模型的分析表明，单营业额动力学数据是一致的两个金属离子结合每个活性位点之间很少或没有积极的协同性。
Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equilibrium and kinetic experiments are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equilibrium fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre-steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre-steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, analysis of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no positive cooperativity between the two metal ions binding each active site.