Kati W M, Johnson K A, Jerva L F, Anderson K S
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
J Biol Chem. 1992 Dec 25;267(36):25988-97.
We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.
我们使用快速瞬态动力学方法，结合特定的合成25/45聚体DNA/RNA和DNA/DNA引物/模板，检测了人类免疫缺陷病毒逆转录酶的RNA依赖性和DNA依赖性聚合酶以及核糖核酸酶H的催化活性。该酶与双链DNA相互作用的解离常数（Kd）值为4.7 nM，与RNA/DNA异源双链体的该值大小相似。对于DNA和RNA模板，均观察到核苷三磷酸掺入的稳态前爆发。对dATP浓度依赖性的爆发速率分析得出，DNA和RNA模板的dATP解离常数（Kd）值分别为4和14 μM，单核苷酸掺入的最大速率（kpol）分别为33和74 s⁻¹。随后的周转受引物/模板从酶上解离速率的限制，双链DNA和RNA/DNA异源双链体的解离速率分别为0.18和0.06 s⁻¹。使用RNA模板对DNA聚合速率和RNA切割速率的分析表明，这两种活性相互独立。聚合速率（4 - 70 s⁻¹）取决于dATP浓度，而在100倍dATP浓度范围（2 - 200 μM）内，RNA切割以10 s⁻¹的恒定速率发生。对单次周转产生的RNA切割产物的检测表明，该酶的聚合酶和核糖核酸酶结构域之间的距离对应于19个RNA/DNA异源双链体碱基，这与最近发表的晶体结构一致（科尔施泰特，L.A.，王，J.，弗里德曼，J.，赖斯，P.A.，和施泰茨T.A.（1992年）《科学》256，1783 - 1790）。对连续合成动力学的分析表明dNTP的初始结合导致DNA从酶上的解离速率加快。进一步研究支持了一种两步dNTP结合机制，即首先形成初始的E.DNA.dNTP复合物，随后形成更稳定的E'.DNA.dNTP复合物。与DNA模板胸腺嘧啶相对掺入错误核苷三磷酸的解离常数（Kd）值，dGTP为1010 μM，dCTP为1240 μM，dTTP为840 μM。相应的最大kpol速率，dGTP为4.8 s⁻¹，dCTP为0.52 s⁻¹，dTTP为0.41 s⁻¹。这些值提供了在该位点对dGTP错掺入的保真度估计为1740，对dCTP为19700，对dTTP为16900。
