Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: Magnesium provides a bridge for ATP to fuel unwinding
Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: Magnesium provides a bridge for ATP to fuel unwinding
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DOI：
10.1016/j.jmb.2006.10.023
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发表时间：
2007-01-26
影响因子：
5.6
通讯作者：
Rypma, Ryan S.
中科院分区：
文献类型：
--
作者：
Frick, David N.;Banik, Sukalyani;Rypma, Ryan S.
本研究探讨了镁离子在丙型肝炎病毒(丙型肝炎病毒)编码的NS3蛋白催化的ATP水解酶和核酸解离中的作用。对不同RNA和镁浓度下的稳态ATP水解率进行了分析，以确定15个解离常数和4个速率常数的值，这15个解离常数描述了产生的酶-金属-ATP-RNA复合体的形成，而4个速率常数描述了可能的酶-ATP复合体对ATP的水解。结合直接结合研究、特异性研究和定点突变的分析，这些值揭示了丙型肝炎病毒解旋酶上只有一个以催化碱基Glu291为中心的ATP结合位点。邻近的残基Asp290与镁离子结合，形成与ATP的桥梁，重新定位活性部位的核苷酸。RNA刺激了水解性，同时降低了酶对三磷酸腺苷、镁和三磷酸镁的亲和力。这里描述的结合方案解释了以前报道的ATP对酶的不寻常调节。丙型肝炎病毒解旋酶与游离镁或游离三磷酸腺苷的结合会与解旋酶运动的真正燃料镁三磷酸腺苷竞争，从而导致较慢的水解和核酸解离。(C)2006爱思唯尔有限公司。保留所有权利。
This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding. (c) 2006 Elsevier Ltd. All rights reserved.