gamma-phosphate-substituted 2'-deoxynucleoside 5'-triphosphates as substrates for DNA polymerases
gamma-phosphate-substituted 2'-deoxynucleoside 5'-triphosphates as substrates for DNA polymerases
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DOI：
10.1074/jbc.271.40.24389
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发表时间：
1996-10-04
影响因子：
4.8
通讯作者：
Krayevsky, AA
中科院分区：
文献类型：
--
作者：
Arzumanov, AA;Semizarov, DG;Krayevsky, AA
合成了几种含有疏水膦酸酯基团而不是γ-磷酸的2 '-脱氧胸苷5'-三磷酸和3 '-叠氮基-2'，3 '-双脱氧胸苷5'-三磷酸类似物，并将其作为人免疫缺陷病毒（HIV）和禽成髓细胞瘤病毒逆转录酶、人胎盘DNA聚合酶α和β以及小牛胸腺末端脱氧核苷酸转移酶的底物进行了评价。它们被逆转录病毒酶有效地整合到DNA链中，但不被哺乳动物酶利用。此外，还合成了dTTP的γ-酯和γ-酰胺衍生物以及3 ′-叠氮基-2 ′，3 ′-双脱氧胸苷5 ′-三磷酸（AZTTP）。它们被证明是研究中的逆转录病毒和哺乳动物酶的底物。dTTP衍生物掺入DNA链的Km值接近dTTP和AZTTP。AZTTP衍生物的Km比dTTP和AZTTP的Km大一个数量级。所获得的结果表明，HIV和禽成髓细胞瘤病毒逆转录酶结合在γ-位上具有庞大取代基的三磷酸片段没有空间障碍。AZTTP中γ-磷酸的修饰增加了HIV逆转录酶抑制对DNA聚合酶α的选择性。γ-甲基膦酸盐和γ-苯基膦酸盐在人血清中的去磷酸化速度远低于AZTTP。此外，它们的疏水性明显高于AZTTP。因此，用γ-膦酸盐替换AZTTP中的γ-磷酸盐显著改变了其对某些细胞DNA聚合酶和血液去磷酸化酶的底物性质，但不改变其对HIV逆转录酶的底物活性。
Several 2'-deoxythymidine 5'-triphosphate and 3'-azido-2',3'-dideoxythymidine 5'-triphosphate analogs containing a hydrophobic phosphonate group instead of the gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases alpha and beta, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some gamma-ester and gamma-amide derivatives of dTTP and 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The K-m values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The K-m for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase alpha. gamma-Methylphosphonate and gamma-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the gamma-phosphate in AZTTP with gamma-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.