Kinetic mechanism of the Mg2+-dependent nucleotidyl transfer catalyzed by T4 DNA and RNA ligases
Kinetic mechanism of the Mg2+-dependent nucleotidyl transfer catalyzed by T4 DNA and RNA ligases
复制标题
DOI：
10.1074/jbc.m109616200
复制
发表时间：
2002-01-18
影响因子：
4.8
通讯作者：
de Vries, S
中科院分区：
文献类型：
--
作者：
Cherepanov, AV;de Vries, S
在没有DNA底物的情况下，用瞬时光吸收光谱和荧光光谱研究了T4DNA和RNA连接酶依赖于镁离子的腺酰化作用。系统地改变了镁离子、三磷酸腺苷和焦磷酸盐的浓度，结果表明核苷酸转移是按双金属离子机制进行的。根据这一机理，只有二镁配位形式的Mg(2)-ATP(0)与酶反应形成共价络合物E-AMP。逆反应(ATP合成)发生在单镁配位的焦磷酸镁和酶(.)镁AMP络合物之间。核苷酸结合率在序列ATP(4-)>；MgATP(2-)>；Mg(2)ATP(0)中降低，表明非共价酶-核苷酸复合体的形成是由静电相互作用驱动的。与RNA连接酶相比，T4DNA连接酶表现出明显更高的ATP结合率和随后的腺化速率，部分原因是它将酶结合的Mg(2)ATP(0)的K-d降低了10倍以上。为了阐明镁在T4DNA和RNA连接酶催化的核苷酸转移中的作用，我们提出了一种过渡态构型，在这种过渡态中，催化的镁离子配位到两个反应的亲核体：形成磷酰胺键的酶的赖氨基部分和ATP的α-β桥氧。
The Mg2+-dependent adenylylation of the T4 DNA and RNA ligases was studied in the absence of a DNA substrate using transient optical absorbance and fluorescence spectroscopy. The concentrations of Mg2+, ATP, and pyrophosphate were systematically varied, and the results led to the conclusion that the nucleotidyl transfer proceeds according to a two-metal ion mechanism. According to this mechanism, only the di-magnesium-coordinated form Mg(2)ATP(0) reacts with the enzyme forming the covalent complex E-AMP. The reverse reaction (ATP synthesis) occurs between the mono-magnesium-coordinated pyrophosphate form MgP2O72- and the enzyme(.)MgAMP complex. The nucleotide binding rate decreases in the sequence ATP(4-) > MgATP(2-) > Mg(2)ATP(0), indicating that the formation of the non-covalent enzyme-nucleotide complex is driven by electrostatic interactions. T4 DNA ligase shows notably higher rates of ATP binding and of subsequent adenylylation compared with RNA ligase, in part because it decreases the K-d of Mg2+ for the enzyme-bound Mg(2)ATP(0) more than 10-fold. To elucidate the role of Mg2+ in the nucleotidyl transfer catalyzed by T4 DNA and RNA ligases, we propose a transition state configuration, in which the catalytic Mg2+ ion coordinates to both reacting nucleophiles: the lysyl moiety of the enzyme that forms the phosphoramidate bond and the alpha-beta-bridging oxygen of ATP.