Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee , 3210 North Cramer Street, Milwaukee, Wisconsin 53211, United States.
Biochemistry. 2013 Sep 10;52(36):6151-9. doi: 10.1021/bi4006495. Epub 2013 Aug 26.
Aurintricarboxylic acid (ATA) is a potent inhibitor of many enzymes needed for cell and virus replication, such as polymerases, helicases, nucleases, and topoisomerases. This study examines how ATA interacts with the helicase encoded by the hepatitis C virus (HCV) and reveals that ATA interferes with both nucleic acid and ATP binding to the enzyme. We show that ATA directly binds HCV helicase to prevent the enzyme from interacting with nucleic acids and to modulate the affinity of HCV helicase for ATP, the fuel for helicase action. Amino acid substitutions in the helicase DNA binding cleft or its ATP binding site alter the ability of ATA to disrupt helicase-DNA interactions. These data, along with molecular modeling results, support the notion that an ATA polymer binds between Arg467 and Glu493 to prevent the helicase from binding either ATP or nucleic acids. We also characterize how ATA affects the kinetics of helicase-catalyzed ATP hydrolysis, and thermodynamic parameters describing the direct interaction between HCV helicase and ATA using microcalorimetry. The thermodynamics of ATA binding to HCV helicase reveal that ATA binding does not mimic nucleic acid binding in that ATA binding is driven by a smaller enthalpy change and an increase in entropy.
金瑞酸（ATA）是一种有效的抑制剂，能够抑制细胞和病毒复制所需的多种酶，如聚合酶、解旋酶、核酸酶和拓扑异构酶。本研究探讨了 ATA 与丙型肝炎病毒（HCV）编码的解旋酶相互作用的方式，并揭示 ATA 会干扰酶与核酸和 ATP 的结合。我们发现 ATA 可以直接与 HCV 解旋酶结合，从而阻止酶与核酸相互作用，并调节 HCV 解旋酶与 ATP 的亲和力，ATP 是解旋酶作用的燃料。解旋酶 DNA 结合凹槽或其 ATP 结合位点中的氨基酸取代会改变 ATA 破坏解旋酶-DNA 相互作用的能力。这些数据以及分子建模结果支持了这样一种观点，即 ATA 聚合物结合在 Arg467 和 Glu493 之间，从而阻止解旋酶结合 ATP 或核酸。我们还利用微量热法研究了 ATA 如何影响解旋酶催化的 ATP 水解动力学以及描述 HCV 解旋酶与 ATA 之间直接相互作用的热力学参数。用 HCV 解旋酶对 ATA 结合的热力学研究表明，ATA 结合并不模拟核酸结合，因为 ATA 结合是由较小的焓变和熵增驱动的。
