STRUCTURAL BASIS FOR THE 3'-5' EXONUCLEASE ACTIVITY OF ESCHERICHIA-COLI DNA-POLYMERASE-I - A 2 METAL-ION MECHANISM
STRUCTURAL BASIS FOR THE 3'-5' EXONUCLEASE ACTIVITY OF ESCHERICHIA-COLI DNA-POLYMERASE-I - A 2 METAL-ION MECHANISM
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DOI：
10.1002/j.1460-2075.1991.tb07917.x
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发表时间：
1991-01-01
期刊：
影响因子：
11.4
通讯作者：
STEITZ, TA
中科院分区：
文献类型：
--
作者：
BEESE, LS;STEITZ, TA
大肠杆菌DNA聚合酶I的大蛋白水解片段（Klenow片段）及其与脱氧核苷单磷酸产物和单链DNA底物的复合物的精细晶体结构提供了编辑3 '-5'核酸外切酶活性位点的详细图像。 这些配合物的结构已被细化到R-因子分别为0.18和0.19，在2.6和3.1埃的分辨率。 与胸苷四核苷酸复合物的复合物显示蛋白质和延伸的四核苷酸之间的许多疏水和氢键相互作用，这解释了该酶使双链体DNA的3'端处的四个核苷酸变性的能力。 这些复合物的结构提供了支持和扩展所提出的用于3 '-5'编辑核酸外切酶反应的两种金属离子机制的细节，所述机制可能对于磷酰基转移酶的大家族是通用的。 对末端核苷酸的磷原子的亲核攻击被假定为通过由一种二价金属激活的氢氧根离子进行，而预期的五配位过渡态和离开的含氧阴离子通过第二种二价金属离子稳定，该第二种二价金属离子与第一种二价金属离子相距3.9埃。 几乎所有方面的前过渡态底物复合物直接看到的结构，只有非常小的变化，磷酸原子的位置需要形成过渡态。
The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5' exonuclease active site. The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6 and 3.1 angstrom resolution respectively. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carried out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 angstrom from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.