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´-[γ-thio]triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5´-[β,γ-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.
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Research Article|
March 25 1999
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
Poul E. JENSEN;
Poul E. JENSEN
1
1Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
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Lucien C. D. GIBSON;
Lucien C. D. GIBSON
2
1Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
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C. Neil HUNTER
C. Neil HUNTER
3
1Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
3To whom correspondence should be addressed (e-mail C.N.Hunter@Sheffield.ac.uk).
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Publisher: Portland Press Ltd
Received:
September 09 1998
Revision Received:
December 17 1998
Accepted:
January 27 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 339 (1): 127–134.
Article history
Received:
September 09 1998
Revision Received:
December 17 1998
Accepted:
January 27 1999
Citation
Poul E. JENSEN, Lucien C. D. GIBSON, C. Neil HUNTER; 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. Biochem J 1 April 1999; 339 (1): 127–134. doi: https://doi.org/10.1042/bj3390127
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