Kinetic parameters for PEP^3-, ADP-Mn and Mn²⁺_free in the absence or presence of 5 mM Fru 1,6-BP or of 5 mM Rib 5-P for <i>Vc</i>IPK and <i>Vc</i>IIPK, respectively.

<p>The data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178673#pone.0178673.g005" target="_blank">Fig 5</a> were fitted (nonlinear regression Origin versión 7.0) to the Hill equation <i>v</i> = <i>V</i>_max* [S]^<i>n</i>+<i>K</i>_{<i>0</i>.<i>5</i>}^<i>n</i><i>+</i> [S] ^<i>n</i>. The mean and standard deviation of three to four experiments are shown.</p

The contribution of two isozymes to the pyruvate kinase activity of <i>Vibrio cholerae</i>: One K⁺-dependent constitutively active and another K⁺-independent with essential allosteric activation

<div><p>In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K⁺-dependent, whereas those with Lys117 were K⁺-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in <i>Vibrio cholerae</i>. The main findings of this work showed that the isozyme with Glu117 is an active K⁺-dependent enzyme. At the same substrate concentration, its <i>V</i>_max in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K⁺-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K⁺-independent enzyme, Mn²⁺ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in <i>Vibrio cholerae</i> have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that <i>Vc</i>IPK and <i>Vc</i>IIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium.</p></div

Kinetic parameters for the monovalent cations in <i>Vc</i>IPK at saturating concentrations of PEP³⁻, ADP-Mg complex and Mg²⁺_free.

<p>The data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178673#pone.0178673.g002" target="_blank">Fig 2A</a> were fitted to the Hill equation <i>v</i> = <i>V</i>_max* [S]^<i>n</i>+<i>K</i>_{<i>0</i>.<i>5</i>}^<i>n</i><i>+</i> [S] ^<i>n</i> (Origin version 7.0). The mean and standard deviation of three experiments are shown.</p

The effect of Fru 1,6-BP in <i>Vc</i>IPK and Rib 5-P in <i>Vc</i>IIPK on the saturation curves for PEP^3-, ADP-Mg and Mg²⁺_free.

<p>In (A, C and E) the effect of Fru 1,6-BP (closed symbols) is shown in <i>Vc</i>IPK and in (B, D and F) the effect of Rib 5-P (open symbols) is shown in <i>Vc</i>IIPK. The concentrations of the allosteric activators were (■,□) 0 mM, (●,○) 0.05 mM and (▲,△) 5 mM. The reaction mixtures contained 25 mM HEPES pH 7.0, 0.2 mM NADH, 8 μg/ml LDH, TMACl in order to maintain the ionic strength at 300–350 mM and with and without 90 mM KCl in <i>Vc</i>IPK and <i>Vc</i>IIPK, respectively. Kinetics for PEP^3- and ADP-Mg were performed under saturating concentrations of the other substrate. The concentrations of Mg²⁺_free for the saturation curves of PEP^3- and ADP-Mg were 2 mM and 15 mM for <i>Vc</i>IPK and <i>Vc</i>IIPK, respectively. For the saturation curves of Mg²⁺_free, the concentration of PEP was varied from 5 to 40 mM and those of ADP-Mg from 3 to 9 mM. Assays were performed at 25°C and the reactions were started by the addition of 0.1 to 5 μg/ml of PK. The data were fitted to the Hill equation. Standard deviation bars of three to four experiments are shown.</p

Effect of various ligands on the intrinsic fluorescence emission spectra at 280 nm, λ_max and the anisotropy of <i>Vc</i>IPK (A) and <i>Vc</i>IIPK (B).

<p>Closed symbols are for <i>Vc</i>IPK and open symbols are for <i>Vc</i>IIPK. The symbols represent: PK + HEPES (■,□), PK + Mg²⁺ (●,○), PK + Mn²⁺ (▲,△), PK + Effector (▼,▽), PK + Effector + Mg²⁺ (◆,◇), PK + Effector + Mn²⁺ (◀,◁). Fluorescence studies were performed at 25°C in mixtures that contained 200 μg ml^-1 of <i>Vc</i>IPK or <i>Vc</i>IIPK in 50 mM HEPES pH 7.0. In <i>Vc</i>IPK, the media contained either 2 mM Mg²⁺, 0.2 mM Mn²⁺ or 5mM of Fru 1,6-BP. In the mixtures that contained Fru 1,6-BP and the divalent cation, the concentration of the effector was 0.5 mM and the concentrations of Mg²⁺ or Mn²⁺ were 2 or 0.2 mM, respectively. In <i>Vc</i>IIPK, the media contained either 30 mM Mg²⁺, 0.5 mM Mn²⁺ or 5 mM Rib 5-P. In the mixtures that contained Rib 5-P and Mg²⁺, the concentrations were 1.5 and 15 mM, respectively. When the effector was added with Mn²⁺, the concentration of Rib 5-P was 0.5 and that of the divalent cation was 0.2 mM. The inset shows the λ_max and the anisotropy values (<i>r</i>). The anisotropy values were calculated at their respective maximal emission.</p

Intracellular concentrations of metabolites in <i>Vibrio cholerae</i> CVD103 grown at stationary phase.

<p>Metabolites were determined by enzymatic coupled assays as described in Experimental Procedures. The mean and standard deviation of three independent cell extracts are shown.</p

Double reciprocal plots from the initial velocity data of the reaction catalyzed by <i>Vc</i>IPK (A and B) and <i>Vc</i>IIPK (C and D).

<p>The reaction mixtures contained 50 mM HEPES pH 7.0, 0.2 mM NADH, 8 μg/ml LDH, 300 mM of ionic strength (TMACl), with 90 mM of KCl and 5 mM of Fru 1,6-BP in <i>Vc</i>IPK and without KCl and 5 mM of Rib 5-P in <i>Vc</i>IIPK. The reciprocals of the concentrations of PEP^3- and ADP-M²⁺ complexes are shown in the abscissas of each graph. The variable fixed concentrations of ADP-Mg in (A) were 0.15 (◀), 0.3 (►), 0.6 (), 0.75 (★) and 1.5 mM () and ADP-Mn in (C) were 0.025 (□), 0.04 (○), 0.06 (△), 0.275 (▽) and 1.3 mM (◇). The variable fixed concentrations of PEP^3- in plot B were 0.06 (◀), 0.16 (►), 0.28 (), 0.4 (★) and 1.21 mM () and in plot D were 0.0408 (□), 0.061 (○), 0.097 (△), 0.28 (▽) and 0.81 mM (◇). In (A and B) the Mg²⁺_free concentration was kept constant at 2 mM and Mn²⁺_free concentration in (C and D) was fixed at 0.35 mM. The reaction was started by the addition of PK, the amounts of PK ranged from 0.1 to 0.5 μg/ml. The fitted data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178673#pone.0178673.t004" target="_blank">Table 4</a>.</p

Kinetic parameters for PEP3-, ADP-Mg and Mg²⁺_free in the absence or presence of 0.05 and 5 mM Fru 1,6-BP for <i>Vc</i>IPK or of 0.05 and 5 mM Rib 5-P for <i>Vc</i>IIPK.

<p>The data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178673#pone.0178673.g004" target="_blank">Fig 4</a> were fitted (nonlinear regression Origin version 7.0) to the Hill equation <i>v</i> = <i>V</i>_max * [S]^<i>n</i>+<i>K</i>_{<i>0</i>.<i>5</i>}^<i>n</i><i>+</i> [S] ^<i>n</i>. The mean and standard deviation of three to four experiments are shown. N. D. not determined.</p

Activation of <i>Vc</i>IPK and <i>Vc</i>IIPK in the presence of 5 mM of different allosteric modulators.

<p>The experimental conditions were 0.5 mM PEP, 6.5 mM ADP-Mg complex and 2 mM Mg²⁺ for <i>Vc</i>IPK (dashed bars) and 5 mM PEP, 6.5 mM ADP-Mg complex and 15 mM Mg²⁺ for <i>Vc</i>IIPK (open bars). The lanes without or with the respective allosteric effector were indicated: control (in the absence of allosteric effector), Rib 5-P, Glc 6-P, Fru 6-P, Fru 1,6-BP and AMP. The activities of the controls were 9 ± 0.12 μmol/min.mg and 1.5 ± 0.11 μmol/min.mg for <i>Vc</i>IPK and <i>Vc</i>IIPK, respectively. Assays were performed at pH 7.0, 300 mM ionic strength, and at 25°C. The reaction was started by addition of 5 μg/ml PK. Standard deviation bars of three experiments are shown.</p

Western blot analysis of <i>Vc</i>IPK (A) and <i>Vc</i>IIPK (B) in <i>Vibrio cholerae</i> cell extracts and cross-reactivity of ployclonal anti-<i>Vc</i>IIPK (C) and anti-<i>Vc</i>IPK (D) against recombinant <i>Vc</i>IPK, <i>Vc</i>IIPK and <i>Vc</i>IIIPK.

<p>Western blot was performed with anti-<i>Vc</i>IPK (A) and anti <i>Vc</i>IIPK (B). MW are the Molecular Weight Standards (Western-C of Biorad) and lanes 2, 3 and 4 are 30 μg of cell extracts of <i>E</i>. <i>coli</i> DH5α, of <i>E</i>. <i>coli</i> PB25 and of <i>V</i>. <i>cholerae</i>, respectively. Lane 5 was loaded with purified recombinant 0.5 μg. of <i>Vc</i>IPK (A) and 1 μg of <i>Vc</i>IIPK (B). In (C) and (D) 0.5 μg of purified recombinant enzymes were loaded. <i>Vc</i>IIIPK was generously given by Dr. Gloria Hernández-Alcántara.</p