The arginine finger of bacteriophage T7 gene 4 helicase: Role in energy coupling
The arginine finger of bacteriophage T7 gene 4 helicase: Role in energy coupling
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DOI：
10.1073/pnas.0400968101
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发表时间：
2004-03-30
影响因子：
11.1
通讯作者：
Richardson, CC
中科院分区：
文献类型：
--
作者：
Crampton, DJ;Guo, SY;Richardson, CC
由噬菌体T7的基因4编码的DNA解旋酶将DNA解旋与dTTP的水解偶联。在原钒酸盐（Vi）存在下的偶联损失表明dTTP的γ-磷酸盐在该机制中起重要作用。六聚体解旋酶的晶体结构显示Arg-522，位于亚基界面，定位为与结合的核苷5'三磷酸的γ-磷酸相互作用。在这方面，它类似于在其他核苷酸水解酶中发现的精氨酸指。当Arg-522被丙氨酸（gp 4-R522 A）或赖氨酸（gp 4-R522 K）取代时，dTTP水解的速率显著降低。改变的蛋白质的dTTT活性不受Vi抑制，表明Vi和基因4蛋白质之间的相互作用丧失。gp 4-R522 A不能解旋DNA，而gp 4-R522 K可以解旋DNA，这与其dTTT活性成比例。然而，gp 4-R522 K不能刺激双链DNA上的T7聚合酶活性。这些发现支持Arg-522残基参与能量偶联机制。
The DNA helicase encoded by gene 4 of bacteriophage T7 couples DNA unwinding to the hydrolysis of dTTP. The loss of coupling in the presence of orthovanadate (Vi) suggests that the gamma-phosphate of dTTP plays an important role in this mechanism. The crystal structure of the hexameric helicase shows Arg-522, located at the subunit interface, positioned to interact with the gamma-phosphate of bound nucleoside 5' triphosphate. In this respect, it is analogous to arginine fingers found in other nucleotide-hydrolyzing enzymes. When Arg-522 is replaced with alanine (gp4-R522A) or lysine (gp4-R522K), the rate of dTTP hydrolysis is significantly decreased. dTTPase activity of the altered proteins is not inhibited by Vi, suggesting the loss of an interaction between Vi and gene 4 protein. gp4-R522A cannot unwind DNA, whereas gp4-R522K does so, proportionate to its dTTPase activity. However, gp4-R522K cannot stimulate T7 polymerase activity on double-stranded DNA. These findings support the involvement of the Arg-522 residue in the energy coupling mechanism.