14 research outputs found
Empirical Valence Bond Simulations of the Chemical Mechanism of ATP to cAMP Conversion by Anthrax Edema Factor
The
two-metal catalysis by the adenylyl cyclase domain of the anthrax
edema factor toxin was simulated using the empirical valence bond
(EVB) quantum mechanical/molecular mechanical approach. These calculations
considered the energetics of the nucleophile deprotonation and the
formation of a new P–O bond in aqueous solution and in the
enzyme–substrate complex present in the crystal structure models
of the reactant and product states of the reaction. Our calculations
support a reaction pathway that involves metal-assisted transfer of
a proton from the nucleophile to the bulk aqueous solution followed
by subsequent formation of an unstable pentavalent intermediate that
decomposes into cAMP and pyrophosphate (PP<sub>i</sub>). This pathway
involves ligand exchange in the first solvation sphere of the catalytic
metal. At 12.9 kcal/mol, the barrier for the last step of the reaction,
the cleavage of the P–O bond to PP<sub>i</sub>, corresponds
to the highest point on the free energy profile for this reaction
pathway. However, this energy is too close to the value of 11.4 kcal/mol
calculated for the barrier of the nucleophilic attack step to reach
a definitive conclusion about the rate-limiting step. The calculated
reaction mechanism is supported by reasonable agreement between the
experimental and calculated catalytic rate constant decrease caused
by the mutation of the active site lysine 346 to arginine
Kinetic parameters of membrane preparations of HEK293 stably overexpressing pGC-A for various nucleotides.
<p>NC activities were analyzed as mentioned in Materials and Methods. Membranes from HEK293 cells overexpressing pGC-A (10–80 µg of protein per tube) were incubated with 2–7,500 µM (XTP/Me<sup>2+</sup>: 2–2,000 µM) NTP/Me<sup>2+</sup> in the presence of 1 µM ANP at 37°C for 5–10 min depending on the analyzed NTP. Apparent s<sub>0.5</sub>, V<sub>max</sub>, and <i>n</i><sub>Hill</sub> represent the means ± SEM of six independent experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-g002" target="_blank">2</a> and are given in alphabetical order of NTPs. Curves were analyzed by nonlinear regression using Prism version 5.0. nd: not detected, nq: not quantified.</p>*<p>only detectable in the presence of 500 µM ATP/Mg<sup>2+</sup>.</p
Inhibition of GC activity by XTP in membrane preparations of HEK293 cells stably overexpressing pGC-A.
<p>Membranes (10 µg protein per tube) was stimulated with 200 µM GTP/Mn<sup>2+</sup> and 1 µM ANP at 37°C for 5 min and increasing concentrations of
XTP/Mn<sup>2+</sup> (2–1,000 µM). Data were best fitted by using a competitive
binding model at one binding site with an IC<sub>50</sub> of 145.3 ± 1.2 µM. Values
based on the means ± SEM of six independent experiments.</p
Analysis of NC activity of ROS membrane preparations.
<p>Membranes (81 µg rhodopsin per tube) were incubated for 5 min at 30°C with 3.5 mM MgCl<sub>2</sub> and 1 mM GTP/Mg<sup>2+</sup>, UTP/Mg<sup>2+</sup> and CTP/Mg<sup>2+</sup>, respectively, and 2 mM EGTA or 2 mM CaCl<sub>2</sub>, as indicated. Reactions were stopped by heating at 95°C for 10 min and analyzed as described in Materials and Methods. Values represent the mean with range of 2–3 independent experiments. Please note the different scales of the y-axes of all panels. A, cNMP formation with membranes; B, NMP formation with membranes; C, control experiments showing contamination of NTP solutions with NMPs.</p
Kinetic data of substrate saturation experiments with CyaA-N and EF.
a<p>data-fit ambiguous,</p>*<p>K<sub>i</sub> for substrate inhibition; N = 3–6; analysis of the data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone.0070223.s004" target="_blank">Figs. S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone.0070223.s005" target="_blank">S5</a>. AC, adenylyl cyclase; CC, cytidylyl cyclase; GC, guanylyl cyclase; IC, inosityl cyclase; TC, thymidinyl cyclase; UC, uridylyl cyclase; XC, xanthosinyl cyclase.</p
Effect of ATP on GC activity in membrane preparations of HEK293 cells stably overexpressing pGC-A.
<p>Membranes (10 µg of protein per tube) were incubated for 25 min at 37°C in the presence of 100 µM GTP/Me<sup>2+</sup> with or without 500 µM ATP/Me<sup>2+</sup> as indicated. Values represent the means ± SEM of three independent experiments. Please note the different scales of the y-axes in both panels.</p
cNMP levels in intact HEK293 cells stably overexpressing pGC-A following stimulation with ANP.
<p>HEK293 cells stably overexpressing pGC-A were seeded in 6-well plates for 24 h with 5⋅10<sup>5</sup> cells per well and stimulated with 1 µM ANP for defined times 10 min after preincubation with IBMX (100 µM). Values of plot A-E are given by means ± SEM of 3–6 independent experiments in µmol/million cells. Please note the different scales of the y-axes in these panels. Plot E: Data points are due to background noise. Dotted lines: lower limit of detection of cIMP, cTMP, and cXMP. ***: p-value ≤0.001.</p
Kinetic analysis of nucleotidyl cyclase activity in membrane preparations of HEK293 cells stably overexpressing pGC-A.
<p>Membranes (10–80 µg protein per tube) were incubated with 2–7,500 µM NTP/Me<sup>2+</sup> (XTP/Me<sup>2+</sup>: 2–2,000 µM) in the presence of 1 µM ANP at 37°C for 5–10 min. Values represent the mean ± SEM of six independent experiments. Except data of plot D, data were fitted using specific binding with Hill slope. Data of plot D were best fitted by substrate inhibition model. Please note the different scales of the x- and y-axes of all panels. *: only detectable in the presence of 500 µM ATP/Mg<sup>2+</sup>. Kinetic parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-t002" target="_blank">Table 2</a>.</p
Parameters for the detection and quantitation of cNMPs and MS standard tenofovir by HLPC-MS/MS.
<p>Protonated molecule mass [M+H]<sup>+</sup>, HPLC retention time, MS/MS fragments of quantifier and qualifier as well as the ratio between quantifier and qualifier for quantitation of cNMP via HPLC-MS/MS.</p
Survival of Sprague Dawley rats (250 to 300 g) treated with single intravenous bolus infusion of LeTx (0.03 mg PA+0.015 mg LF) or EdTx (0.15 mg PA+0.075 mg EF) and monitored for two weeks.
<p>These are the same doses used for telemetry and ECHO experiments. N = 12 for each experiment. Kaplan-Meier graphs prepared.</p