中文摘要：

E.coli热诱导赖氨酰-tRNA合成酶（LysU,EC 6.1.1.6）是高效的Ap4A/Ap3A合成酶,已知反应模式为双重动态过程：2ATP→Ap4A＋2Pi→Ap3A＋3Pi。为进一步研究LysU＂中间物可逆＂催化模型,表达纯化了LysU蛋白并验证结构稳定性,构建了二腺苷多磷酸产物检测系统并分离了各阶段催化产物,观察了AMPPCP和AMPCPP阻断Ap3A/ADP合成的反应。圆二色光谱和荧光光谱扫描证明纯化后的LysU蛋白结构完整。LysU首先催化ATP合成83%的Ap4A,接着可逆生成67%的Ap3A。实验中发现,Ap3A并非LysU二腺苷多磷酸催化反应的终产物,Ap3A可继续逆生成80%的ADP。以AMPPCP或AMPCPP代替ATP为起始底物,发现无Ap3A转化ADP反应。上述结果证明LysU具有三重催化活性：2ATP→Ap4A＋2Pi→Ap3A＋3Pi→2ADP＋2Pi,符合＂磷酸捕获机制＂催化模型：活化的赖氨酰-腺苷中间物捕获核苷酸或磷酸小分子,形成对应的二腺苷多磷酸化合物。这些研究结果可为阐明不同形式功能性腺苷酸衍生物间的相互转化提供更多的信息,有助于进一步认识功能性腺苷酸分子在生命活动中的作用。

英文摘要：

E.coli heat-inducible lysyl-tRNA synthetase（LysU,EC6.1.1.6） is known to be a highly efficient Ap4A/Ap3A synthase with dual catalytic activity： 2ATP →Ap4A＋2Pi→Ap3A＋3Pi.To get better known on the intermediate reversible model of LysU,LysU was purified and confirmed its structural stability by circular dichroism and intrinsic fluorescence spectrum.An easy-fast ion-exchange chromatography technique was set up and products were sequentially separated for mass spectrometry.Results demonstrated that LysU initially produce 83% Ap4A from ATP,and then turn over Ap4A into 67% Ap3A.However,LysU was accidentally discovered can consecutively catalyze Ap3A into 85% ADP as the final product.By using AMPPCP and AMPCPP as ATP surrogate,the Ap3A/ADP reaction was blocked.Mechanistic investigations proved that LysU is triple catalytic activity enzyme： 2ATP →Ap4A＋2Pi→Ap3A＋3Pi→2ADP＋2Pi.Therefore,LysU catalyze the formation of Ap4A/Ap3A/ADP via phosphate-based trap mechanism which is either phosphate-base as inorganic phosphate as added ADP,or nucleotide as ADP as added Ap3A.