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.

Coupled ATPase-adenylate kinase activity in ABC transporters

“…(ref. 39), cannot be observed. Instead, two new resonances corresponding to the βP and γP positions of ATP-βSγS appear proving that the protein follows reaction scheme (2), a reverse adenylate kinase reaction.…”

“…Here, for probing its catalytic activity within the lipid bilayer, we utilize real-time 31 P-MAS NMR to monitor substrate turnover by recording progress curves for all compounds involved. This approach has been shown to offer unique insight into membrane protein catalysed reactions involving, for example, ATP hydrolysis and phosphoryl group transfers39. Furthermore, 1 H– 31 P cross-polarization experiments are used for identifying transporter-bound nucleotide species.…”

“…The 31 P isotope is the ideal probe for fast, label-free and noninvasive observation of reactions because of its large chemical shift dispersion, its high sensitivity and 100% natural abundance as NMR-active nuclei. As demonstrated for diaclygylcerol kinase, coupled reactions at and within the lipid bilayer can be monitored directly and simultaneously39. Based on single-point mutations introduced in the Walker A, Signature, D-loop, Q-loop and Walker-B motifs of the MsbA NBDs, a model is proposed in which the canonical nucleotide-binding site, needed for ATP hydrolysis, together with a postulated second site forms the AK reaction centre.…”

Molecular properties of diacylglycerol kinase-epsilon in relation to function

“…The DgkA from Gram positive bacteria is primarily an undecaprenol kinase, while DgkA from Gram negative bacteria is a DAG-kinase (Jerga et al, 2007). DgkA from the Gram negative bacteria, Escherichia coli, also catalyzes the transfer of phosphate from ATP not only to lipid species, but also to molecules of water and glycerol (Ullrich et al, 2011;Prodeus et al, 2013). A study using 31 P NMR to detect ATP hydrolysis and DAG phosphorylation was used to investigate ATPase activity of human DGKe and determine if water competes with DAG as an acceptor of the g phosphate of ATP as is seen with DgkA (Prodeus et al, 2013).…”

BSH-CP based 3D solid-state NMR experiments for protein resonance assignment

“…Magic-angle spinning (MAS) solid-state NMR (ssNMR) has evolved in the past decade into a powerful technique to determine structure and dynamics of insoluble proteins (Goldbourt 2013;Knight et al 2012;Sengupta et al 2012) and protein complexes (Mainz et al 2013), such as amyloid fibrils (Tycko 2011;Wasmer et al 2008), membrane proteins (Hong et al 2012;Ullrich et al 2011) and oligomeric assemblies (Jehle et al 2010;Loquet et al 2012). This became possible in part due to the availability of higher external magnetic fields and the development of twodimensional (2D) and three-dimensional (3D) homonuclear and heteronuclear correlation experiments.…”