Tan C K, Civil R, Mian A M, So A G, Downey K M
Department of Medicine, University of Miami School of Medicine, Florida 33101.
Biochemistry. 1991 May 21;30(20):4831-5. doi: 10.1021/bi00234a001.
The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.
已对叠氮胸苷一磷酸（AZTMP）和其他核苷5'-单磷酸对重组HIV逆转录酶的RNA依赖性DNA聚合酶及核糖核酸酶H活性的影响进行了研究。在以氯化镁作为二价阳离子激活剂时，这两种活性对毫摩尔浓度的AZTMP抑制均敏感。用锰离子（Mn2+）替代镁离子（Mg2+）可显著增强AZTMP对核糖核酸酶H活性的抑制作用，使半数抑制浓度（IC50）从5毫摩尔降至0.05毫摩尔。相比之下，Mn2+不会改变RNA依赖性DNA聚合酶活性对AZTMP抑制的敏感性。增加底物聚腺苷酸/聚脱氧胸苷酸（poly(A)/poly(dT)）的浓度可逆转AZTMP对核糖核酸酶H活性的抑制作用，这表明AZTMP可能与底物竞争结合核糖核酸酶H的活性位点。在存在Mg2+的情况下，其他核苷5'-单磷酸不会抑制核糖核酸酶H。然而，在存在Mn2+时，与异源双链底物的DNA链互补的脱氧和双脱氧核苷5'-单磷酸具有一定抑制作用。在Mg2+或Mn2+存在下，RNA依赖性DNA聚合酶活性会受到AZTMP和双脱氧胸苷一磷酸（ddTMP）的轻微抑制，用Mn2+替代Mg2+也会导致双脱氧腺苷一磷酸（ddAMP）产生抑制作用。天然存在的核糖或脱氧核糖核苷5'-单磷酸在浓度高达5毫摩尔时无抑制作用。由于叠氮胸苷三磷酸（AZTTP）在纳摩尔浓度下就能抑制HIV逆转录酶的RNA依赖性DNA聚合酶活性，因此AZTMP对该活性的抑制作用不太可能在AZT的抗病毒效应中发挥重要作用。然而，AZTMP对核糖核酸酶H活性的抑制作用（在体内可达到毫摩尔浓度）可能是病毒对AZT敏感的部分原因。
