Divita G, Müller B, Immendörfer U, Gautel M, Rittinger K, Restle T, Goody R S
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Biochemistry. 1993 Aug 10;32(31):7966-71. doi: 10.1021/bi00082a018.
Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3'-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial association (k(ass) = ca. 5 x 10(8) M-1 s-1) followed by a slow isomerization step (k = ca. 0.5 s-1). These (forward) rate constants are increased in the presence of a non-nucleoside inhibitor (S-TIBO) of HIV-1 reverse transcriptase, while the reverse rate constant for the second step is decreased, leading to an increase in affinity between the enzyme and primer/template by a factor of at least 10 when S-TIBO is bound. The results are discussed in terms of present knowledge of the structure of reverse transcriptase.
来自HIV-1和HIV-2的逆转录酶的内在蛋白质荧光为监测这些酶与引物/模板双链分子的相互作用提供了一个灵敏的信号。发现18/36-mer DNA/DNA双链体的解离常数（Kd）值在几纳摩尔范围内（HIV-2的酶的Kd值比HIV-1的酶的Kd值高约3倍）。结合引物/模板时诱导的蛋白质荧光猝灭，以及与在引物3'-端含有荧光核苷酸的引物/模板相互作用时外在荧光的增加，被用于研究与HIV-1逆转录酶相互作用的动力学。结果可以用两步结合模型来解释，即快速的扩散限制初始缔合（缔合速率常数k(ass) = 约5×10(8) M-1 s-1），随后是缓慢的异构化步骤（速率常数k = 约0.5 s-1）。在HIV-1逆转录酶的非核苷抑制剂（S-TIBO）存在下，这些（正向）速率常数增加，而第二步的逆向速率常数降低，当S-TIBO结合时，导致酶与引物/模板之间的亲和力增加至少10倍。根据目前对逆转录酶结构的了解对结果进行了讨论。
