The Mg(2+)-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 Mg(2+), 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 MgP(2)O(7)(2-) 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 Mg(2+) for the enzyme-bound Mg(2)ATP(0) more than 10-fold. To elucidate the role of Mg(2+) in the nucleotidyl transfer catalyzed by T4 DNA and RNA ligases, we propose a transition state configuration, in which the catalytic Mg(2+) ion coordinates to both reacting nucleophilesthe lysyl moiety of the enzyme that forms the phosphoramidate bond and the alpha-beta-bridging oxygen of ATP.

译文

Mg(2)依赖的T4 DNA和RNA连接酶的腺苷酸化在不存在DNA底物的情况下使用瞬时光吸收和荧光光谱进行了研究。 Mg(2),ATP和焦磷酸盐的浓度发生了系统性的变化,结果得出的结论是,核苷酸基转移是根据两种金属离子机理进行的。根据此机制,只有二价镁配体形式Mg(2)ATP(0)与酶反应形成共价复合物E.AMP。逆反应(ATP合成)发生在单镁配位的焦磷酸盐形式MgP(2)O(7)(2-)与酶MgAMP复合物之间。核苷酸结合率降低顺序ATP(4-)> MgATP(2-)> Mg(2)ATP(0),表明非共价酶与核苷酸复合物的形成是由静电相互作用驱动的。与RNA连接酶相比,T4 DNA连接酶显示出显着更高的ATP结合率和随后的腺苷酸化,部分原因是它使结合酶的Mg(2)ATP(0)的Mg(2)的K(d)降低10以上。 -折叠。为了阐明Mg(2)在T4 DNA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,其中Mg(2)催化离子与两个亲核试剂反应形成酶的赖氨酰部分氨基磷酸酯键和ATP的α-β桥接氧。

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