Harris D, Yadav P N, Pandey V N
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark 07103, USA.
Biochemistry. 1998 Jul 7;37(27):9630-40. doi: 10.1021/bi980549z.
Tyr183 is a constituent of the highly conserved YXDD motif common to all retroviral reverse transcriptases. The two aspartates in this motif are the crucial members of the catalytic carboxylate triad while residue X, which in the case of HIV-1 RT is Met184, is implicated in dNTP substrate recognition and fidelity of DNA synthesis. In an attempt to understand the function of Tyr183 in the catalytic mechanism, we generated mutants of this residue (Y183F and Y183A) and subjected them to in-depth analysis. The efficiency of reverse transcription of natural U5-PBS HIV-1 RNA template was severely impaired by both the conservative and nonconservative substitutions. The major defect identified was at the level of dNTP binding as determined by a 20-80-fold increase in the Km for the dNTP substrate on both homopolymeric and heteropolymeric RNA and DNA templates. A significant reduction in processivity of DNA synthesis by these mutants was also noted. However, the fidelity of DNA synthesis by the Y183F and Y183A mutants was increased significantly compared to the wild-type enzyme. Interestingly, the reduction in the polymerase activity due to single substitution of Tyr to Phe in the YMDD motif is compensated by a second substitution of Met to Val in the same motif, herein referred to as the FVDD. The loss of dNTP binding as well as decreased processivity of DNA synthesis exhibited by the Y183F mutant was also compensated by mutation at the second site. Curiously, the double mutant did not exhibit any synergistic effect in regard to fidelity of DNA synthesis as might be expected since both the single mutations (Y183F, M184V) exhibited enhanced fidelity compared to the wild-type enzyme. These data implicate Tyr183 and Met184 as important constituents of the dNTP-binding pocket. We propose a model which suggests that subtle structural changes due to mutation in the flexible beta9-beta10 loop region at the active site of the molecule influence the enzyme activity and substrate recognition.
酪氨酸183是所有逆转录病毒逆转录酶共有的高度保守的YXDD基序的组成部分。该基序中的两个天冬氨酸是催化性羧酸盐三联体的关键成员，而残基X（在HIV-1逆转录酶中为甲硫氨酸184）与脱氧核苷三磷酸（dNTP）底物识别及DNA合成保真度有关。为了理解酪氨酸183在催化机制中的功能，我们构建了该残基的突变体（Y183F和Y183A）并对其进行深入分析。天然U5-PBS HIV-1 RNA模板的逆转录效率在保守和非保守取代时均受到严重损害。通过对同聚和异聚RNA及DNA模板上dNTP底物的米氏常数（Km）增加20至80倍确定，主要缺陷在于dNTP结合水平。还注意到这些突变体的DNA合成持续能力显著降低。然而，与野生型酶相比，Y183F和Y183A突变体的DNA合成保真度显著提高。有趣的是，YMDD基序中酪氨酸单取代为苯丙氨酸导致的聚合酶活性降低，可通过同一基序中甲硫氨酸第二次取代为缬氨酸来补偿，此处称为FVDD。Y183F突变体表现出的dNTP结合丧失及DNA合成持续能力降低也可通过第二位点的突变得到补偿。奇怪的是，该双突变体在DNA合成保真度方面未表现出预期的协同效应，因为与野生型酶相比，两个单突变（Y183F、M184V）均表现出增强的保真度。这些数据表明酪氨酸183和甲硫氨酸184是dNTP结合口袋的重要组成部分。我们提出一个模型，表明分子活性位点处柔性β9-β10环区域的突变引起的细微结构变化会影响酶活性和底物识别。
