Poliovirus RNA-dependent RNA polymerase (3Dpol):: Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+
Poliovirus RNA-dependent RNA polymerase (3Dpol):: Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+
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DOI：
10.1021/bi035213q
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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;Gohara, DW;Cameron, CE
在聚合酶催化反应中使用 Mn2+ 代替 Mg2+ 作为二价阳离子辅助因子通常会降低底物选择和掺入保真度的严格性。我们已经解决了在 Mn2+ 存在下由脊髓灰质炎病毒 (3D(pol)) 的 RNA 依赖性 RNA 聚合酶催化的单核苷酸掺入的完整动力学机制。核苷酸掺入的单个循环期间所采用的步骤与Mg2+存在时所采用的步骤相同，并且包括核苷酸结合后的构象变化步骤，以实现聚合酶-引物/模板-核苷酸复合物的催化能力。在 Mn2+ 存在的情况下，构象变化步骤是酶特异性的主要决定因素，磷酰基转移似乎是核苷酸掺入的唯一限速步骤，并且磷酰基转移速率对于所有核苷酸都是相同的：正确和错误。由于磷酰基转移是 Mn2+ 存在下的限速步骤，因此可以确定该系统中的最大硫代磷酸酯效应在 8-11 范围内。该信息允许在 Mg2+ 存在的情况下进一步询问核苷酸选择过程，突出显示该阳离子允许酶使用磷酰基转移步骤进行核苷酸选择的能力。当使用不正确的底物时，Mn2+ 无法支持磷酰基转移效率的降低，这是在存在该辅因子的情况下观察到的保真度损失的主要原因。我们认为构象变化涉及将结合核苷酸的三磷酸部分重新定向为允许结合催化所需的第二种金属离子的构象。在 Mg2+ 存在的情况下，这种构象需要与酶相互作用，从而在尝试掺入不正确的核苷酸时导致催化效率降低。当在 Mg2+ 存在的情况下结合不正确的核苷酸时，辅因子结合位点与结合的 Mn2+ 的偶然相互作用可能会通过补偿用于调节催化效率的相互作用损失来降低保真度。
The use of Mn2+ as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg2+ often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D(pol)) in the presence of Mn2+. The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg2+ and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn2+, the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn2+, it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg2+, highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn2+ to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg2+, this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn2+ may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg2+.