Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4
Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4
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DOI：
10.1074/jbc.m003357200
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发表时间：
2000-11-24
影响因子：
4.8
通讯作者：
Rao, VB
中科院分区：
文献类型：
--
作者：
Leffers, G;Rao, VB
二十面体噬菌体中的双链DNA包装被认为是由包装“机器”驱动的，该包装“机器”由门蛋白和组装在空前头壳的独特门顶点处的两个包装/末端酶蛋白构成。虽然ATP水解显然是主要的驱动力，但包装机械的哪一个组件起着易位ATP酶的作用还没有阐明。有证据表明，大包装亚基是一个强有力的候选人易位ATP酶。我们已经构建了新的噬菌体T7启动子控制下的T4末端酶重组和过表达的包装/末端酶蛋白gp 16和gp 17在各种配置。六组氨酸标记的包装蛋白通过Ni 2 +-琼脂糖层析纯化至接近均一，并且显示出对包装DNA的高度活性。包装大亚基gp 17而不是小亚基gp 16表现出ATP酶活性。尽管gp 16缺乏ATP酶活性，但它使gp 17相关的ATP酶活性增强>50倍。gp 16增强是特异性的，并且是由于ATP水解的催化速率增加。磷酸化的gp 17被证明在低催化速率的条件下，但不是在gp 16的存在下的高催化速率。这些数据与包装机组装后弱ATP酶转化为高催化能力的易位ATP酶的假设一致。
Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell. Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated. Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations. The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni2+-agarose chromatography and were shown to be highly active for packaging DNA in, vitro. The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity. Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold, The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis. A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16. The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.