Functional analysis of amino acid residues constituting the dNTP binding pocket of HIV-1 reverse transcriptase
Functional analysis of amino acid residues constituting the dNTP binding pocket of HIV-1 reverse transcriptase
复制标题
DOI：
10.1074/jbc.273.50.33624
复制
发表时间：
1998-12-11
影响因子：
4.8
通讯作者：
Pandey, VN
中科院分区：
文献类型：
--
作者：
Harris, D;Kaushik, N;Pandey, VN
为了了解构成人类免疫缺陷病毒1型逆转录酶的dNTP结合口袋的残基的功能含义，我们在位置65、72、113、115、151、183、184和219处进行了定点诱变，并检查了所得突变酶的生化特性和对RNA和DNA模板的核苷酸选择性。位置65、115、183、184和219处的突变对聚合酶活性具有可忽略的至中等的影响，而位置72和151处的Ala取代以及位置113处的Ala或Glu取代严重损害了酶的聚合酶功能。K219 A、Y115 F和Q151 M突变体对保真度没有影响; Y183 A、Y183 F、K65 A和Q151 N突变体在RNA和DNA模板上都表现出更高的保真度，而Y115 A在DNA模板上的选择性较低。对酶-模板引物-dNTP三元复合物的三维模型的分析表明，残基Tyr-183、Lys-65和Gln-151可能凭借其与构成口袋的dNTP、模板、引物和其他相邻残基的多重相互作用而对dNTP结合口袋的柔性产生影响。正确核苷酸与不正确核苷酸的募集可能是该口袋的柔性的函数。与柔性口袋相比，相对刚性的口袋将提供更大的严格性，导致DNA合成的更高保真度。用具有降低的相互作用能力的残基取代具有多重相互作用的残基将改变袋的内部几何形状，从而直接影响保真度。
In order to understand the functional implication of residues constituting the dNTP-binding pocket of human immunodeficiency virus type 1 reverse transcriptase, we performed site-directed mutagenesis at positions 65, 72, 113, 115, 151, 183, 184, and 219, and the resulting mutant enzymes were examined for their biochemical properties and nucleotide selectivity on RNA and DNA templates. Mutations at positions 65, 115, 183, 184, and 219 had negligible to moderate influence on the polymerase activity, while Ala substitution at positions 72 and 151 as well as substitution with Ala or Glu at position 113 severely impaired the polymerase function of the enzyme. The K219A, Y115F, and Q151M mutants had no influence on the fidelity; Y183A, Y183F, K65A, and Q151N mutants exhibited higher fidelity on both RNA and DNA templates, while Y115A was less error-prone selectively on a DNA template. Analysis of the three-dimensional model of the enzyme-template primer-dNTP ternary complex suggests that residues Tyr-183, Lys-65, and Gln-151 may have impact on the flexibility of the dNTP-binding pocket by virtue of their multiple interactions with the dNTP, template, primer, and other neighboring residues constituting the pocket. Recruitment of the correct versus incorrect nucleotides may be a function of the flexibility of this pocket. A relatively rigid pocket would provide greater stringency, resulting in higher fidelity of DNA synthesis in contrast to a flexible pocket. Substitution of a residue having multiple interactions with a residue having reduced interaction capability will alter the internal geometry of the pocket, thus directly influencing the fidelity.