Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli.
Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli.
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DOI：
10.1016/s0021-9258(18)42461-1
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发表时间：
1992-04
期刊：
影响因子：
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通讯作者：
A. Polesky;M. Dahlberg;S. Benkovic;N. Grindley;C. M. Joyce
中科院分区：
文献类型：
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作者：
A. Polesky;M. Dahlberg;S. Benkovic;N. Grindley;C. M. Joyce
为了继续我们对 DNA 聚合酶 I 的 Klenow 片段的聚合酶活性位点的诱变研究，我们表征了在 Asp705、Glu710 和 Glu883 处进行取代的新突变体，并进一步研究了残基 Arg668、Gln849 和 Asp882 中的突变，这些突变在我们之前的研究中表现出强烈的 Kcat 效应。为了确定每个突变蛋白的反应步骤是限速的，我们测量了 α-硫代取代的 dNTP 对反应速率的影响。一组突变体显示出显着的硫元素效应，而第二组突变体，如野生型 Klenow 片段，不受硫代磷酸酯取代的影响。与早期数据一致，这些结果表明，在酶-DNA-dNTP 三元复合物的形成和磷酸二酯键形成的完成之间，存在两个动力学上不同的步骤，其中只有一个对硫取代敏感。用克列诺片段的突变衍生物获得的相当复杂的元素效应数据更符合元素效应是过渡态之一的空间冲突的结果，而不是基于硫和氧之间的电负性差异的解释。数据表明 Asp882 的侧链参与了所提出的空间冲突，并且 Gln849 和 Glu883 参与了反应的硫敏感步骤。根据我们的结果以及与其他磷酰基转移酶的比较，讨论了聚合酶反应的可能机制。
To continue our mutagenesis study of the polymerase active site of the Klenow fragment of DNA polymerase I, we have characterized new mutants with substitutions at Asp705, Glu710, and Glu883, and have investigated further the mutations in residues Arg668, Gln849, and Asp882, which showed strong Kcat effects in our previous study. To determine which step of the reaction is rate-limiting for each mutant protein, we measured the effect on the reaction rate of an alpha-thio-substituted dNTP. One group of mutants showed a substantial sulfur elemental effect, while a second group, like wild-type Klenow fragment, was unaffected by the phosphorothioate substitution. Consistent with earlier data, these results imply that, between formation of the enzyme-DNA-dNTP ternary complex and completion of phosphodiester bond formation, there are two kinetically distinct steps, only one of which is sensitive to sulfur substitution. The rather complex elemental effect data obtained with the mutant derivatives of Klenow fragment are more consistent with the elemental effect being the result of a steric clash in one of the transition states than with explanations based on electronegativity differences between sulfur and oxygen. The data suggest that the side chain of Asp882 is involved in the proposed steric clash, and that Gln849 and Glu883 participate in the sulfur-sensitive step of the reaction. Based on our results, and comparisons with other phosphoryl transfer enzymes, possible mechanisms for the polymerase reaction are discussed.