Poliovirus RNA-dependent RNA polymerase (3Dpol):: Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2+
Poliovirus RNA-dependent RNA polymerase (3Dpol):: Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2+
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DOI：
10.1021/bi035212y
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发表时间：
2004-05-11
期刊：
影响因子：
2.9
通讯作者：
Cameron, CE
中科院分区：
文献类型：
--
作者：
Arnold, JJ;Cameron, CE
我们已经解决了脊髓灰质炎病毒RNA依赖RNA聚合酶3D（pol）催化正确核苷酸整合的完整动力学机制。磷酸化转移步骤的两侧是两个异构化步骤。第一个构象变化可能与结合的核苷酸的三磷酸部分的重新定向有关，第二个构象变化可能是酶的易位到下一轮核苷酸结合的位置。观察到的3D（pol）结合核苷酸的速率常数（86 s(-1)）由第一次构象变化（300 s(-1)）和磷酸化转移（520 s(-1)）的速率常数决定。“活化”三元配合物稳定性的变化与观察到的核苷酸修饰引起的掺入速率常数的变化最相关。除UTP外，核苷酸的K-d值至少比相应核苷酸的细胞浓度低10倍。我们的数据预测，过渡突变应该以1/15000的频率发生，翻转突变应该以小于1/150000的频率发生，2'-脱氧核糖核苷酸与正确碱基的结合应该以1/7500的频率发生。总之，这些数据支持这样的结论，即3D（pol）实际上与外切酶缺陷的复制性DNA聚合酶一样可靠。我们讨论了这项工作对开发RNA依赖性RNA聚合酶抑制剂作为抗病毒药物的意义。
We have solved the complete kinetic mechanism for correct nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol). The phosphoryl-transfer step is flanked by two isomerization steps. The first conformational change may be related to reorientation of the triphosphate moiety of the bound nucleotide, and the second conformational change may be translocation of the enzyme into position for the next round of nucleotide incorporation. The observed rate constant for nucleotide incorporation by 3D(pol) (86 s(-1)) is dictated by the rate constants for both the first conformational change (300 s(-1)) and phosphoryl transfer (520 s(-1)). Changes in the stability of the "activated" ternary complex correlate best with changes in the observed rate constant for incorporation resulting from modification of the nucleotide. With the exception of UTP, the K-d values for nucleotides are at least 10-fold lower than the cellular concentration of the corresponding nucleotide. Our data predict that transition mutations should occur at a frequency of 1/15000, transversion mutations should occur at a frequency of less than 1/150000, and incorporation of a 2'-deoxyribonucleotide with a correct base should occur at a frequency 1/7500. Together, these data support the conclusion that 3D(pol) is actually as faithful as an exonuclease-deficient, replicative DNA polymerase. We discuss the implications of this work on the development of RNA-dependent RNA polymerase inhibitors for use as antiviral agents.