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首页> 外文期刊>Biochemistry >BIOCHEMICAL ANALYSIS OF CATALYTICALLY CRUCIAL ASPARTATE MUTANTS OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REVERSE TRANSCRIPTASE
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BIOCHEMICAL ANALYSIS OF CATALYTICALLY CRUCIAL ASPARTATE MUTANTS OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REVERSE TRANSCRIPTASE

机译：人体免疫缺陷病毒1型逆转录酶催化十字花青酸盐突变体的生物化学分析

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摘要

In order to clarify the role(s) of the individual member of the carboxylate triad in the catalytic mechanism of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we carried out site-directed mutagenesis of D185, D186, and D110, followed by the extensive characterization of the properties of the individual mutant enzymes. We find that all three residues participate at or prior to the chemical step of bond formation. The incorporation pattern seen with phosphorothioate analogs of dNTP on both RNA-DNA and DNA-DNA template-primers indicated that D186 may be the residue that coordinates with the alpha-phosphate group of dNTP in the transition-state ternary complex. Further support for the role assigned to D186 was obtained by examination of the ability of the individual carboxylate mutants to catalyze the reverse of the polymerase reaction (pyrophosphorolysis). Mutants of D185 exhibited near-normal pyrophosphorolysis activity, while those of D186 were completely devoid of this activity. Thus, D185 appears to participate only in the forward reaction, probably required for the generation of nucleophile by interacting with the 3'-OH of the primer terminus, while D186 seems to be involved in both the forward and the reverse reactions, presumably by participating in the pentavalent intermediate transition state. Lack of any elemental effects during polymerization with mutant enzymes of residue D110, together with their inability to catalyze pyrophosphorolysis, suggest its probable participation in the metal-coordinated binding to the beta-gamma-phosphate of dNTP or PPi in the forward and reverse reactions. respectively. A molecular model of the ternary complex based on these results is also presented.
机译：为了阐明羧酸盐三联体的单个成员在人类免疫缺陷病毒1型（HIV-1）逆转录酶催化机制中的作用，我们进行了D185，D186和D110的定点诱变，其次是各个突变酶的特性的广泛表征。我们发现，所有三个残基都在化学键形成步骤之前或之前参与。在RNA-DNA和DNA-DNA模板引物上都用dNTP的硫代磷酸酯类似物观察到的掺入模式表明，D186可能是与过渡态三元复合物中的dNTP的α-磷酸酯基团配位的残基。通过检查单个羧酸盐突变体催化聚合酶反应（焦磷酸解）逆向反应的能力，进一步确定了D186的作用。 D185的突变体表现出接近正常的焦磷酸解活性，而D186的突变体完全没有这种活性。因此，D185似乎仅参与正向反应，可能是通过与引物末端的3'-OH相互作用产生亲核试剂所需的，而D186似乎既参与正向反应又参与了反向反应，大概是通过参与在五价中间过渡态。与残基D110的突变酶聚合期间缺乏任何元素效应，以及它们无法催化焦磷酸解，表明其可能在正向和反向反应中与金属配位的dNTP或PPi的β-γ-磷酸结合。分别。还基于这些结果给出了三元复合物的分子模型。

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