Double-reciprocal plots for steady-state kinetics of <i>Mt</i>SK using either ATP (A) or SKH (B) as the variable substrate.

<p>Each curve represents varied-fixed levels of the co-substrate, ranging from 37 to 4800 µM for SKH and from 9 to 1200 µM to ATP.</p

Thermodynamics parameters of formation of binary complexes between <i>Mt</i>SK and substrate(s) or product(s).

<p>K_D represents the equilibrium dissociation constant, ΔH is the binding enthalpy, ΔS is the binding entropy, ΔG is the Free Gibbs energy, and –TΔS is the negative term for temperature (in Kelvin) times binding entropy.</p

<i>Mt</i>SK:SKH thermodynamic binding parameters as a function of temperature (A) and binding enthalpy as a function of buffer ionization enthalpy at pH 7.6 (B).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918#pone-0061918-g007" target="_blank">Figure 7A</a> shows the ΔH (filled circles), -TΔS (open circles) and ΔG (inverted filled triangles) dependence on a temperature ranging from 10 to 40°C, which permits determination of a ΔC_p value of −320 (±16) cal mol⁻¹ K⁻¹. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918#pone-0061918-g007" target="_blank">Figure 7B</a> shows the dependence of observed enthalpy on buffer ionization enthalpy (ΔH_ion) at 25 °C. Data fitting to Eq. 6 yielded a value of −0.47 for N_H+ (number of protons exchanged during the binding process) and −2.2 kcal mol⁻¹ for ΔH_int (intrinsic enthalpy).</p

Hydrophobic and vibrational thermodynamic parameters of <i>Mt</i>SK:SKH binary complex formation.

<p>ΔC_p represents the heat capacity; the H and V subscripts represent, respectively, the hydrophobic and vibrational contributions.</p

Fluorescence spectroscopy of the equilibrium binding of SKH to <i>Mt</i>SK, measuring the quench in intrinsic protein fluorescence upon ligand binding andp plotting the relative fluorescence change as a function of SKH concentration.

<p>Fluorescence spectroscopy of the equilibrium binding of SKH to <i>Mt</i>SK, measuring the quench in intrinsic protein fluorescence upon ligand binding andp plotting the relative fluorescence change as a function of SKH concentration.</p

Steady-state kinetics constants of Shikimate Kinases (SKs) from different organisms.

1<p>Results described here.</p>2<p>adapted from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918#pone.0061918-Gu1" target="_blank">[43]</a>.</p>3<p>SK from <i>Erwinia chrysanthemi</i>. (values taken from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918#pone.0061918-Krell1" target="_blank">[54]</a>).</p

Shikimate Kinase catalyzed phosphoryl transfer from ATP to C3 hydroxyl group of shikimate (SKH), yielding shikimate 3-phosphate (S3P) and ADP.

<p>Shikimate Kinase catalyzed phosphoryl transfer from ATP to C3 hydroxyl group of shikimate (SKH), yielding shikimate 3-phosphate (S3P) and ADP.</p

12% SDS-PAGE analysis of pooled fractions of Mt<i>SK</i> for each purification step.

<p>Lane 1, Protein Molecular Weight Marker (Fermentas); lane 2, soluble <i>E. coli</i> BL21 (DE3) [pET-23a(+)::aroK] extract; lane 3, Soluble proteins after 10 mM MgCl₂ precipitation step; lane 4, Phenyl Sepharose 16/10; and lane 5, Sephacryl S-100 HR.</p