title: Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR.
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 (31)P 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.
authors.affiliation: Institute for Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany.
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Ullrich
authors.forename: Ute A
authors.initials: UA
authors.lastname: Hellmich
authors.forename: Stefan
authors.initials: S
authors.lastname: Ullrich
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D019852:Diacylglycerol Kinase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010712:Phosphatidic Acids / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding
chemical_list: D010712:Phosphatidic Acids; D000255:Adenosine Triphosphate; D019852:Diacylglycerol Kinase