Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR

Journal name:
Nature Chemical Biology
Volume:
7,
Pages:
263–270
Year published:
DOI:
doi:10.1038/nchembio.543
Received
Accepted
Published online

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.

At a glance

Figures

  1. Time-resolved 31P MAS NMR is used to follow the phosphorylation of DOG, which is catalyzed by DGK reconstituted in DOPC bilayers at the expense of ATP.
    Figure 1: Time-resolved 31P MAS NMR is used to follow the phosphorylation of DOG, which is catalyzed by DGK reconstituted in DOPC bilayers at the expense of ATP.

    (a) Direct polarization spectra yield information on the hydrolysis of ATP and phosphorylation of the lipid substrate DOG, regardless of its compartmentalization: the resonances of αP-ATP (−11.0 p.p.m.), βP-ATP (−19.5 p.p.m.) and γP-ATP (−6.0 p.p.m.) decrease, those of αP-ADP (−10.5 p.p.m.), βP-ADP (−6.7 p.p.m.), free Pi (−0.8 p.p.m.) and DOG-PA (0.1 p.p.m.) increase over time. (b) Cross polarization experiments select for immobile species, therefore visualizing only the membrane-bound part of the reaction, the DOG phosphorylation. (c) Complete deconvolution of a and b yields progress curves (inset), which can be approximated by monoexponential functions, except for Pi, which shows a lag phase. For DOG phosphorylation, direct polarization and cross polarization experiments yield rates of 7.94(3) × 10−3 min−1 and 8.53(4) × 10−3 min−1, respectively. As a control, the average of all progress curves is plotted and found to be constant during the time course of the experiment. The MAS rotor (70 μl) contained 0.15 μmol DGK reconstituted in DOPC (1:120) 2 μmol MgATP and 2 μmol DOG. Data were recorded at 293 K. See Methods for further information.

  2. Progress curves showing DGK's enzymatic activity extracted from time-resolved 31P-DP MAS spectra recorded without and with DBG present.
    Figure 2: Progress curves showing DGK's enzymatic activity extracted from time-resolved 31P-DP MAS spectra recorded without and with DBG present.

    (a,b) Individual progress curves for αP-, βP- and γP-ATP, αP- and βP-ADP, DBG-PA and Pi can be approximated well by monoexponential functions (see Supplementary Table 2). DGK's ATPase activity is stimulated approximately fivefold upon addition of DBG. The gray bar in a corresponds to the time range in b. The buildup of free Pi in b is greatly diminished because the γ-phosphate of ATP is transferred to DBG to yield DBG-PA. As a control, the time course of the average of all integrals is plotted in black in a and b and is found to be almost constant. The MAS rotor (70 μl) contained 0.15 μmol DGK reconstituted 1:120 in DOPC, 2 μmol MgATP and 0.175 μmol DBG. Data were recorded at 303 K. See Methods section and Supplementary Table 2 for further details.

  3. Kinetic analysis of rates and stoichiometry of the reaction catalyzed by DGK revealing cooperativity and efficiency.
    Figure 3: Kinetic analysis of rates and stoichiometry of the reaction catalyzed by DGK revealing cooperativity and efficiency.

    (a) The rates for DGK-catalyzed ATP hydrolysis and DBG phosphorylation obtained from time-resolved 31P-MAS NMR data sets are plotted in a semi-log and linear fashion as a function of DBG concentration (40 mM corresponds to 13.5 mol%). Fitting the ATP data with a Hill equation returns a Hill coefficient of n = 3.2 ± 0.4 and a = 2.8 ± 0.3 mM (1.1 ± 0.1 mol%). For comparison, curves for n = 2.5 and 4.0 are shown (dotted line). For DBG, a value of n = 1.4 ± 0.4 and = 4.7 ± 0.3 mM (1.8 ± 0.1 mol%) was found. Dotted lines correspond to n = 1.0 and 1.8. The ratio between the ATP hydrolysis and DBG phosphorylation rates yields a stoichiometry of 1:1.5 (ATP/DBG-PA). The stoichiometry can also be obtained from ATP and DBP-PA peak integrals from the first and the last 31P spectra acquired during the time course of DGK's enzymatic reaction. (b) Molar amounts of consumed ATP and generated DBG-PA under DBG titration are shown. At full saturation, a ratio of ATP/DBG-PA of approximately 1.5:1 is obtained. The same experimental parameters as in Figure 2 apply. See Methods section for further details.

  4. The effects of Vi and ATPγS on DGK activity allow mechanistic insights.
    Figure 4: The effects of Vi and ATPγS on DGK activity allow mechanistic insights.

    (a) Modulation of DGK's ATP hydrolysis rates upon addition of Vi (DGK/Vi = 1:2.5). The ATPase activity of DGK was reduced to about 35% (gray) but could still be stimulated by addition of DBG. Substrate phosphorylation, as monitored by 31P-MAS NMR spectra, did not occur in presence of Vi. (b) Modulation of DGK's ATP hydrolysis rates when ATP is replaced by ATPγS. The hydrolysis rate is not stimulated by DBG but about tenfold enhanced compared to the nonstimulated case. With ATPγS, DBG phosphorylation is observed in the NMR spectra. (c) Time-resolved 31P-MAS NMR spectra of DGK (0.75 mg) in the presence of 2 μmol ATPγS . The signals of αP- (−11.1 p.p.m.), βP- (−20.7 p.p.m.) and γP-ATPγS (36.4 p.p.m.) decay with time. Simultaneously, the resonances for αP- and βP-ADP appear at −10.6 p.p.m. and −7.5 p.p.m., respectively. The generation of free s-Pi can be observed through the increase of its resonance at 44.5 p.p.m. (d) Same experimental conditions as in c but with stimulation by 0.45 μmol DBG. The buildup of the resonance of the thiophosphorylated lipid product DBG-s-PA is observed at 47.3 p.p.m. The inset shows spectra acquired at the beginning (black line) and in the end (red line) of the reaction. DGK transfers thiophosphate of ATPγS (36.4 p.p.m.) to DBG resulting in thiophosphorylated DBG and s-Pi. Both resonances are found in comparison to their nonthiolated counterparts at 0.8 p.p.m. and 44.5 p.p.m. for Pi and s-Pi and at 0.6 p.p.m. and 47.3 p.p.m. for DBG-PA and DBG-s-PA.

  5. Three-dimensional surface model of DGK trimer.
    Figure 5: Three-dimensional surface model of DGK trimer.

    The top view of DGK trimer (PDB ID: 2KDC)22 shows the three intertwined monomers I–III (upper right). The side view shows the DGK trimer in the membrane. The residues, which showed sensitivity to ATP (blue), lipid (red) or both (yellow) are accentuated. Our results favor a mechanistic model with different binding sites for hydrophilic ATP and membrane-bound hydrophobic diacylglycerol. The two substrates can bind independently from one another in a random-equilibrium mechanism. DAG stimulates the ATP hydrolysis rate in a cooperative way with a Hill coefficient of 3.2 ± 0.4, indicating a concerted mechanism of the protein trimer. The DAG-stimulated ATPase activity and DAG phosphorylation could be associated with different DAG binding sites (red). A high degree of intersubunit cross-talk is required as indicated by the intertwined structural model. The γ-phosphate of ATP is transferred to the DAG in a direct phosphoryl transfer mechanism via a transfer site (indicated in yellow) that can be blocked by transition state phosphate analogs such as Vi.

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Author information

  1. These authors contributed equally to this work.

    • Sandra J Ullrich &
    • Ute A Hellmich

Affiliations

  1. Institute for Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany.

    • Sandra J Ullrich,
    • Ute A Hellmich &
    • Clemens Glaubitz
  2. Centre for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main, Germany.

    • Sandra J Ullrich,
    • Ute A Hellmich &
    • Clemens Glaubitz
  3. Institute for Organic Chemistry and Chemical Biology, Goethe University, Frankfurt am Main, Germany.

    • Stefan Ullrich

Contributions

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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    Supplementary Methods, Supplementary Figures 1–4 and Supplementary Tables 1 & 2

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