The simultaneous observation of interdependent reactions within different phases, as catalyzed by membrane bound enzymes, is still a challenging task. One such enzyme, the E. 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 Mg*ATP to membrane-bound diacylglycerol. We demonstrate that time-resolved 31P-MAS NMR offers the unique possibility to simultaneously and directly detect both ATP hydrolysis as well as diacylgylcerol 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 analogues. Our data yield conclusions about inter-subunit cooperativity, reaction stoichiometries, phosphoryl transfer mechanism and are discussed in the context of known structural data.
- Ullrich SJ, Hellmich UA, Ullrich S, Glaubitz C. Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR. Nat Chem Biol.;7:263 (2011).