Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase.
Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase.
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谷氨酰胺 151 参与 HIV-1 逆转录酶的底物 dNTP 结合功能。
DOI：
10.1021/bi00021a036
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Modak,MJ
中科院分区：
文献类型：
--
作者：
Sarafianos,SG;Pandey,VN;Kaushik,N;Modak,MJ
修订稿于 1995 年 3 月 17 日收到® 摘要：为了确定 Glnl51 在 HIV-1 RT 聚合酶功能中的作用，我们通过用保守残基 (Q151N) 和非保守残基 (Q151A) 取代该残基，对该残基进行了定点诱变。 Q151N 表现出与野生型酶类似的特性，而 Q151A 的聚合酶活性严重受损。 Q151A 突变体在使用 RNA [poly (rC) 和 poly (rA)] 模板时表现出 15-100 倍的 kCM 降低，而使用 DNA [poly (dC)] 模板时仅观察到 5 倍的降低。最有趣的是，Q151A 突变体对 dNTP 底物的亲和力在 RNA 模板下保持不变，但在 DNA 模板下 Km 显着增加。根据光亲和交联判断，Q151A 对 DNA 的结合亲和力保持不变。然而，与野生型酶不同，Q151A突变体未能催化共价固定模板引物的引物末端上的核苷酸基转移酶反应。该酶显示出从 Mg2+ 到 Mn2+ 的二价阳离子偏好发生了深刻变化。这些结果强烈暗示 HIV-1 RT 的 Q151 参与底物 dNTP 结合功能，并可能参与随后的化学（催化）步骤。该突变的影响似乎是通过 p66 催化亚基的 Q151 产生的，因为 p66wn/P51qi5ia 保留了野生型动力学常数和核苷酸转移酶活性。相比之下，p66Q151A/p51 wt 与 Q151A（两个亚基均发生突变）无法区分。三元复合物模型（酶-模板引物和 dNTP）已被用来推断 Q151 可能与底物的碱基部分以及与 HIV-1 RT 活性位点内存在的残基 Arg72 相互作用的可能模式。对特定抑制剂具有抗性的人类免疫缺陷病毒 (HIV-1) 毒株的迅速出现，使控制获得性免疫缺陷综合征传播的努力受挫。对针对 HIV-1 逆转录酶 (HIV-1 RT) 1 的抑制剂的抗性被认为是由于该酶的突变所致 (Larder & Kemp, 1989; Larder et al., 1989a, b, 1991)。因此，必须彻底了解 HIV-1 RT 的催化机制。 HIV-1RT的X射线结构
Revised Manuscript Received March 17, 1995® abstract: In order to define the role of Glnl51 in the polymerase function of HIV-1 RT, we carried out site-directed mutagenesis of this residue by substituting it with a conserved (Q151N) and a nonconserved residue (Q151A). Q151N exhibited propertiesanalogous to those of the wild-type enzyme, while Q151A has severely impaired polymerase activity. The Q151A mutant exhibited a 15—100-fold reduction in kCM with RNA [poly (rC) and poly (rA)] templates, while only a 5-fold reduction could be seen with the DNA [poly (dC)] template. Most interestingly, the affinity of the Q151A mutant for dNTP substrate remained unchanged with RNA templates, but a significant increase in Km was noted with the DNA template. The binding affinity of Q151A for DNA remained unchanged, as judged by photoaffinity cross-linking. However, unlike the wild-type enzyme, the Q151A mutant failed to catalyze the nucleotidyl transferase reaction onto the primer terminus of the covalently immobilized template-primer. The enzyme showed profoundly altered divalent cation preference from Mg2+ to Mn2+. These results stronglyimplicate Q151 of HIV-1 RT in the substrate dNTP binding function and possiblyin the following chemical (catalytic) step. The effects of the mutation seem to be through Q151 of the p66 catalytic subunit, as p66wn/P51qi5ia retains the wild-type kinetic constants and nucleotidyl transferase activity. In contrast, p66Q151A/p51 wt is indistinguishable from Q151A (mutated in both subunits). A model of the ternary complex (enzyme—template-primer and dNTP) has been used to infer the possible mode bywhich Q151 may interact with the base moiety of the substrate as well as with Arg72, a residue present within the active site of HIV-1 RT.The rapid emergence of human immunodeficiency virus (HIV-1) strains resistant to specific inhibitors has frustrated the efforts to control the spread of the acquiredimmunodeficiency syndrome. The resistance to inhibitors directed at the reverse transcriptase of HIV-1 (HIV-1 RT) 1 is thought to be due to mutationsin thisenzyme (Larder & Kemp, 1989; Larder et al., 1989a, b, 1991). It is thereforeimperative that a thorough understanding of the catalytic mechanism of HIV-1 RT be achieved. The X-ray structure of HIV-1RT