Abstract
The simultaneous observation of interdependent reactions within different phases as catalyzed by membrane-bound enzymes is still a challenging task. One such enzyme, the Escherichia coli integral membrane protein diacylglycerol kinase (DGK), is a key player in lipid regulation. It catalyzes the generation of phosphatidic acid within the membrane through the transfer of the γ-phosphate from soluble MgATP to membrane-bound diacylglycerol. We demonstrate that time-resolved 31P magic angle spinning NMR offers a unique opportunity to simultaneously and directly detect both ATP hydrolysis and diacylglycerol phosphorylation. This experiment demonstrates that solid-state NMR provides a general approach for the kinetic analysis of coupled reactions at the membrane interface regardless of their compartmentalization. The enzymatic activity of DGK was probed with different lipid substrates as well as ATP analogs. Our data yield conclusions about intersubunit cooperativity, reaction stoichiometries and phosphoryl transfer mechanism and are discussed in the context of known structural data.
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Acknowledgements
We are grateful to J. Becker-Baldus, L. Buchner, S. Hölper, C. Kaiser, N. Pfleger and L. Reggie for technical assistance. Plasmids were kindly provided by C.R. Sanders (Vanderbilt University). We thank D. O'Donovan and J.J. Lopez for initial help with data analysis. We thank M. Göbel for enabling access to his laboratory for substrate synthesis and B. Ludwig for helpful discussions. M. Lorch is acknowledged for valuable comments to the manuscript. The work was supported by the Lipid Signalling Forschungszentrum Frankfurt (LiFF) and by SFB 807 “Membrane Transport.” Further support by the EU consortium Lipid-PRISM and the Cluster of Excellence Macromolecular Complexes Frankfurt is acknowledged. S.J.U. is grateful for a stipend from the “Studienstiftung des deutschen Volkes.”
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S.J.U., U.A.H. and C.G. designed the project. S.J.U. carried out protein preparation. S.J.U. and S.U. synthesized DBG. S.J.U. and U.A.H. performed NMR experiments. S.J.U. carried out data analysis. S.J.U., U.A.H. and C.G. carried out data interpretation and wrote the manuscript. C.G. supervised the project.
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Ullrich, S., Hellmich, U., Ullrich, S. et al. Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR. Nat Chem Biol 7, 263–270 (2011). https://doi.org/10.1038/nchembio.543
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DOI: https://doi.org/10.1038/nchembio.543
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