Kaushik N, Rege N, Yadav P N, Sarafianos S G, Modak M J, Pandey V N
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark 07103, USA.
Biochemistry. 1996 Sep 10;35(36):11536-46. doi: 10.1021/bi960364x.
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.
为了阐明羧酸三联体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，我们对D185、D186和D110进行了定点诱变，随后对各个突变酶的性质进行了广泛表征。我们发现，所有这三个残基都在键形成的化学步骤或之前参与其中。在RNA-DNA和DNA-DNA模板引物上使用dNTP的硫代磷酸酯类似物观察到的掺入模式表明，D186可能是在过渡态三元复合物中与dNTP的α-磷酸基团配位的残基。通过检查单个羧酸盐突变体催化聚合酶反应逆转（焦磷酸解）的能力，进一步支持了赋予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的突变体则完全没有这种活性。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相互作用来产生亲核试剂所必需的，而D186似乎参与正向和反向反应，大概是通过参与五价中间体过渡态。与D110残基的突变酶聚合过程中缺乏任何元素效应，以及它们无法催化焦磷酸解，表明其可能分别在正向和反向反应中参与与dNTP或PPi的β-γ-磷酸的金属配位结合。还基于这些结果提出了三元复合物的分子模型。
