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首页> 外文期刊>Biochemistry >Dynamic, thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase
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Dynamic, thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase

机译：人体免疫缺陷病毒1种整合酶识别和转化DNA的动态，热力学和动力学和动力学基础

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Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn2+, the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide similar to8 orders of magnitude in the affinity (DeltaG(o) approximate to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to similar to1.5 orders of magnitude (DeltaG(o) approximate to -2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides. [References: 39]
机译：逆转录病毒整合酶（In）和长末端重复之间的特异性相互作用是将病毒DNA插入宿主基因组中。为了定量表征衬底特异性的决定因素，我们使用了基于配体复杂性逐步增加的方法。这允许估计每个核苷酸的相对贡献从寡核苷酸与寡核苷酸的总亲和力。使用热力学模型分析HIV-1整合酶与特异性（含有来自LTR的序列）的相互作用或非特异性寡核苷酸。整合酶通过与它们的碱叠加的弱触点叠加，更重要的是与寡核苷酸相互作用，更重要的是，具有核苷酸磷酸基团。所有这些结构部件都以与CA二核苷酸的保守3'-末端GT的主要贡献结合的自由能量的组合方式贡献。与抑制in in催化的反应的非特异性寡核苷酸相反，特异性寡核苷酸增强了活性，这可能由于序列特异性配体对寡聚形式的动态平衡的影响而产生。然而，通过与MN2 +孵育的孵育开始活化后，特定的寡核苷酸也能够抑制加工反应。我们发现，在DNA中的非特异性相互作用在亲和力（Δ近似至-10.3kcal / mol）中提供了类似的数量级（Δtag（o）），而基板的特定核苷酸的相对贡献对应于类似的-1.5次幅度（deltag（o）近似到-2.0 kcal / mol）。在in和特定DNA之间形成迈克利斯综合体本身不能占酶特异性的主要贡献，这是k（猫）术语;在从非特异性到特异性寡核苷酸的转变时，速率增加了超过5个级。 [参考：39]

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