Steady-state NTPase activity of Dengue virus NS3: number of catalytic sites, nucleotide specificity and activation by ssRNA.
Steady-state NTPase activity of Dengue virus NS3: number of catalytic sites, nucleotide specificity and activation by ssRNA.
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DOI：
10.1371/journal.pone.0058508
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Kaufman SB
中科院分区：
文献类型：
--
作者：
Incicco JJ;Gebhard LG;González-Lebrero RM;Gamarnik AV;Kaufman SB
登革病毒非结构蛋白3（NS 3）在核苷三磷酸水解产生的自由能的驱动下解旋双链RNA。本文首次系统和定量表征了DENV NS 3的稳态NTR活性及其与ssRNA的相互作用。获得了ATP、GTP、CTP和UTP的底物曲线，并且这些核苷酸的特异性顺序-以比率（kcat/KM）评估-为GTPATPCTP UTP，这表明NS 3在不同NTP之间具有差的区分能力。四种底物之间的竞争实验表明，它们都是在一个相同的催化位点的酶水解。采用poly（A）和poly（C）研究ssRNA对NS 3 ATP酶活性的影响。这两种RNA分子产生的周转率常数（kcat）增加10倍，ATP的表观亲和力（KM）降低100倍。当[RNA碱基]/[NS_3]比值在0 ~ 20之间时，poly（A）和poly（C）的增加均抑制ATP酶活性。利用大配体（NS 3）与无限长的一维均匀晶格（RNA）结合的理论，我们检验了抑制是NS 3分子沿着RNA晶格拥挤的结果的假设。最后，我们讨论了为什么这一假设是一致的想法，ATP酶催化循环是紧密耦合到NS 3解旋酶沿着RNA的运动。
Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (kcat/KM)- was GTPATPCTP UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and 20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.
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影响因子：
14.9
作者：
Gorbalenya AE;Koonin EV;Donchenko AP;Blinov VM
通讯作者：
Blinov VM
DOI：
10.3390/v3091739
发表时间：
2011-09
期刊：
Viruses
影响因子：
--
作者：
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Gamarnik AV
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作者：
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通讯作者：
Fu, JL
影响因子：
3.7
作者：
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通讯作者：
Padmanabhan, R