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首页> 外文期刊>Biochemistry >ROLE OF ADENOSINE 5'-TRIPHOSPHATE HYDROLYSIS IN THE ASSEMBLY OF THE BACTERIOPHAGE T4 DNA REPLICATION HOLOENZYME COMPLEX
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ROLE OF ADENOSINE 5'-TRIPHOSPHATE HYDROLYSIS IN THE ASSEMBLY OF THE BACTERIOPHAGE T4 DNA REPLICATION HOLOENZYME COMPLEX

机译：腺嘌呤5'-磷酸的水解在噬菌体T4 DNA复制全酶复合物中的作用

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摘要

Steady-state and pre-steady-state rates of ATP hydrolysis by the 44/62 accessory protein were determined to elucidate the role of ATP hydrolysis in bacteriophage T4 holoenzyme complex formation. Steady-state ATPase measurements of the 44/62 protein under various combinations of 45 protein, DNA substrate, and T4 exo(-) polymerase indicate that although the 44/62 protein synergistically hydrolyzes ATP in the presence of 45 protein and DNA substrate, the ATPase activity of 44/62 is diminished substantially upon the formation of the holoenzyme complex. The decrease in activity is primarily in k(cat) while the K-m for ATP is changed unsubstantially by the various combinations. Data suggest that the decrease in the rate of ATP hydrolysis upon the addition of T4 exo(-) polymerase in the presence of 45 protein and DNA substrate is due to formation of a stable holoenzyme complex consisting of only the 45 protein and T4 exo(-) polymerase in a 1:1 ratio. The 44/62 protein acts catalytically to load 45 protein onto the DNA substrate and does not remain a component of the holoenzyme complex. Pre-steady-state kinetic analysis of the ATP hydrolysis reaction catalyzed by the 44/62 protein loading the 45 protein onto the DNA substrate in the absence or presence of polymerase is biphasic, in which a burst in ATP hydrolysis precedes the steady-state rate of ATP hydrolysis. An identical burst in ATP consumption is obtained under either condition, indicating that ATP hydrolysis is not required to load polymerase into the holoenzyme complex. The data suggest one turnover of ATP at each of the four ATPase active sites of the 44/62 protein per 45 protein loaded. ATP hydrolysis by the 44/62 protein under conditions of holoenzyme complex formation is the rate-limiting step in holoenzyme complex formation. The process of holoenzyme formation appears to be identical for leading and lagging strand synthesis.
机译：确定44/62辅助蛋白的ATP水解的稳态和稳态前速率，以阐明ATP水解在噬菌体T4全酶复合物形成中的作用。在45种蛋白质，DNA底物和T4 exo（-）聚合酶的各种组合下，对44/62蛋白的稳态ATPase测量表明，尽管在存在45种蛋白质和DNA底物的情况下，44/62蛋白可以协同水解ATP，但是当全酶复合物形成时，44/62的ATP酶活性大大降低。活性的降低主要是在k（cat）中，而ATP的K-m因各种组合而无明显变化。数据表明，在存在45种蛋白质和DNA底物的情况下，添加T4 exo（-）聚合酶后ATP水解速率降低是由于形成了仅由45种蛋白质和T4 exo（-）组成的稳定的全酶复合物。 ）聚合酶的比例为1：1。 44/62蛋白具有催化作用，可将45种蛋白加载到DNA底物上，而不是完整酶复合物的组成部分。在不存在或存在聚合酶的情况下，由44/62蛋白质将45种蛋白质上样到DNA底物上而催化的ATP水解反应的稳态前动力学分析是双相的，其中ATP水解的爆发先于稳态速率ATP水解。在任一条件下，ATP消耗量都相同，这表明不需要ATP水解即可将聚合酶加载到全酶复合物中。数据表明，每装载45个蛋白质，在44/62蛋白的四个ATPase活性位点中的每个ATP转换一次。在完整酶复合物形成的条件下，44/62蛋白的ATP水解是完整酶复合物形成的限速步骤。全酶形成的过程对于前导链和滞后链的合成似乎是相同的。

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