ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803:: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits
ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803:: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits
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DOI：
10.1042/0264-6021:3390127
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发表时间：
1999-04-01
影响因子：
4.1
通讯作者：
Hunter, CN
中科院分区：
文献类型：
--
作者：
Jensen, PE;Gibson, LCD;Hunter, CN
镁原卟啉IX螯合酶(镁螯合酶)催化的镁离子插入原卟啉IX被认为是一个两步反应，包括活化和镁离子的络合。激活步骤需要ATP和两个亚基，Chli和Chld(分别为I和D)，据推测，这一步骤会导致I-D-ATP复合体的形成。在接下来的步骤中，将镁离子插入到原卟啉中，除了需要ATP激活的I-D复合体外，还需要ATP和第三亚单位H。在本研究中，我们研究了蓝藻聚球藻PCC6803镁螯合酶的I和D亚基之间的相互作用。我们证明了I-D复合体的纯化，并证明了ATP和Mg2+是形成这种复合体的绝对要求，可能就是MgATP。然而，ATP可以被缓慢水解的类似物，5‘-[伽马-硫代]三磷酸取代，在一定程度上，也可以被ADP和不可水解性的腺苷5’-[β，伽马亚胺]三磷酸取代，所有这些都表明，ATP的水解不是形成CHLI-CHLD络合物所必需的。用灵敏的连续测定法测定了镁离子络合过程中的ATPase活性，发现ATP水解酶的最大速率与镁离子的最大插入速率一致。ATP的水解率取决于决定镁离子络合速率的因素，如增加H亚基的浓度和原卟啉的浓度。因此，三磷酸腺苷的水解已被确定为螯合步骤的绝对要求。单独检测时，I亚基具有较强的ATPase活性，而D亚基没有检测到活性，当I和D亚基联合检测时，I亚基的ATPase活性受到抑制。
Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation. The activation step requires ATP and two of the subunits, ChlI and ChlD (I and D respectively), and it has been speculated that this step results in the formation of an I-D-ATP complex. The subsequent step, in which Mg2+ is inserted into protoporphyrin, also requires ATP and the third subunit, H, in addition to ATP-activated I-D complex. In the present study, we examine the interaction of the I and D subunits of the Mg chelatase from the cyanobacterium Synechocystis PCC 6803. We demonstrate the purification of an I-D complex, and show that ATP and Mg2+ are absolute requirements for the formation of this complex, probably as MgATP. However, ATP may be replaced by the slowly hydrolysable analogue, adenosine 5'-[gamma-thio]triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5'-[beta,gamma-imido]triphosphate, all of which suggests that ATP hydrolysis is not necessary for the formation of the ChlI-ChlD complex. A sensitive continuous assay was used to detect ATPase activity during Mg2+ chelation, and it was found that the maximum rate of ATP hydrolysis coincided with the maximum rate of Mg2+ insertion. The rate of ATP hydrolysis depended on factors that determined the rate of Mg2+ chelation, such as increasing the concentration of the H subunit and the concentration of protoporphyrin. Thus ATP hydrolysis has been identified as an absolute requirement for the chelation step. The I subunit possessed strong ATPase activity when assayed on its own: whereas the D subunit had no detectable activity, and when the I and D subunits were assayed in combination, the ATPase activity of the I subunit was repressed.