Davis W R, Tomsho J, Nikam S, Cook E M, Somand D, Peliska J A
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Biochemistry. 2000 Nov 21;39(46):14279-91. doi: 10.1021/bi0015764.
DNA strand transfer reactions occur twice during retroviral reverse transcription catalyzed by HIV-1 reverse transcriptase. The 4-chlorophenylhydrazone of mesoxalic acid (CPHM) was found to be an inhibitor of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Using a model strand transfer assay system described previously [Davis, W. R., et al. (1998) Biochemistry 37, 14213-14221], the mechanism of CPHM inhibition of DNA strand transfer has been characterized. CPHM was found to target the RNase H activity of HIV-1 reverse transcriptase. DNA polymerase activity was not significantly affected by CPHM; however, it did inhibit the polymerase-independent RNase H activity with an IC(50) of 2.2 microM. In the absence of DNA synthesis, CPHM appears to interfere with the translocation, or repositioning, of RT on the RNA.DNA template duplex, a step required for efficient RNA hydrolysis by RNase H. Enzyme inhibition by CPHM was found to be highly specific for HIV-1 reverse transcriptase; little or no inhibition of DNA strand transfer or DNA polymerase activity was observed with MLV or AMV reverse transcriptase, T7 DNA polymerase, or DNA polymerase I. Examination of additional 4-chlorophenylhydrazones showed that the dicarboxylic acid moiety of CPHM is essential for activity, suggesting its important role for enzyme binding. Consistent with the role of the dicarboxylic acid in inhibitor function, Mg(2+) was found to chelate directly to CPHM with a K(d) of 2.4 mM. Together, these studies suggest that the inhibitor may function by binding to enzyme-bound divalent metal cofactors.
在由HIV-1逆转录酶催化的逆转录病毒逆转录过程中，DNA链转移反应会发生两次。发现中草酸4-氯苯腙（CPHM）是HIV-1逆转录酶催化的DNA链转移反应的抑制剂。使用先前描述的模型链转移分析系统[戴维斯，W.R.等人（1998年）《生物化学》37卷，14213 - 14221页]，对CPHM抑制DNA链转移的机制进行了表征。发现CPHM靶向HIV-1逆转录酶的核糖核酸酶H活性。DNA聚合酶活性未受到CPHM的显著影响；然而，它确实以2.2微摩尔的半数抑制浓度（IC50）抑制了不依赖聚合酶的核糖核酸酶H活性。在没有DNA合成的情况下，CPHM似乎会干扰逆转录酶（RT）在RNA·DNA模板双链体上的易位或重新定位，这是核糖核酸酶H有效水解RNA所需的步骤。发现CPHM对酶的抑制作用对HIV-1逆转录酶具有高度特异性；用莫洛尼氏鼠白血病病毒（MLV）或禽成髓细胞瘤病毒（AMV）逆转录酶、T7 DNA聚合酶或DNA聚合酶I未观察到对DNA链转移或DNA聚合酶活性的抑制作用或仅有轻微抑制作用。对其他4-氯苯腙的研究表明，CPHM的二羧酸部分对活性至关重要，这表明其在酶结合中具有重要作用。与二羧酸在抑制剂功能中的作用一致，发现镁离子（Mg2+）以2.4毫摩尔的解离常数（Kd）直接与CPHM螯合。总之，这些研究表明该抑制剂可能通过与酶结合的二价金属辅因子结合而起作用。
