Kinetic and Structural Investigations into the Allosteric and pH Effect on the Substrate Specificity of Human Epithelial 15-Lipoxygenase‑2

Lipoxygenases, important enzymes in inflammation, can regulate their substrate specificity by allosteric interactions with their own hydroperoxide products. In this work, addition of both 13-(<i>S</i>)-hydroxy-(9<i>Z</i>,11<i>E</i>)-octadecadienoic acid [13-(<i>S</i>)-HODE] and 13-(<i>S</i>)-hydroperoxy-(6<i>Z</i>,9<i>Z</i>,11<i>E</i>)-octadecatrienoic acid to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the <i>k</i>_cat/<i>K</i>_M substrate specificity ratio of arachidonic acid (AA) and γ-linolenic acid (GLA) by 4-fold. 13-(<i>S</i>)-HODE achieves this change by activating <i>k</i>_cat/<i>K</i>_M^AA but inhibiting <i>k</i>_cat/<i>K</i>_M^GLA, which indicates that the allosteric structural changes at the active site discriminate between the length and unsaturation differences of AA and GLA to achieve opposite kinetic effects. The substrate specificity ratio is further increased, 11-fold in total, with an increase in pH, suggesting mechanistic differences between the pH and allosteric effects. Interestingly, the loss of the PLAT domain affects substrate specificity but does not eliminate the allosteric properties of 15-LOX-2, indicating that the allosteric site is located in the catalytic domain. However, the removal of the PLAT domain does change the magnitude of the allosteric effect. These data suggest that the PLAT domain moderates the communication pathway between the allosteric and catalytic sites, thus affecting substrate specificity. These results are discussed in the context of protein dimerization and other structural changes

Determination of IC₅₀ values for ketoconazole and ketaminazole with CaCYP51 and HsCYP51.

<p>CYP51 reconstitution assays (0.5-ml total volume) containing 1 µM CaCYP51 (A) or 0.3 µM HsCYP51 (B) were performed as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a>. Ketoconazole (solid circles) and ketaminazole (hollow circles) concentrations were varied from 0 to 4 µM for CaCYP51 and up to 190 µM for HsCYP51 with the DMSO concentration kept constant at 1% (vol/vol). Mean values from two replicates are shown along with associated standard deviation bars. Relative velocities of 1.0 were equivalent to 1.04 and 2.69 nmoles 14α-demetylated lanosterol produced per minute per nmole CYP51 (min⁻¹) for CaCYP51 and HsCYP51, respectively.</p

Whole human blood activity profile of representative analogues^a.

a<p>The ELISA absorption-based inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section. Compounds were assayed at 10 µM.</p

5-LOX IC₅₀ values of representative analogues (µM), with errors in brackets.

<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section.</p

Buffer conditions for IC₅₀ assays, with constant substrate concentration and varying inhibitor concentration^a.

a<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section.</p

Selectivity profile of representative analogues (µM), with errors in parentheses^a.

a<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section.</p>b<p>N/D = Not determined.</p

IC₅₀ values of dual anti-fungal, anti-inflammatory inhibitors (µM), with error in parentheses.

<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section. N/D = Not determined.</p

Representative analogues evaluated for pseudoperoxidase activity and IC₅₀ potency (µM), with errors in brackets.

<p>The UV-based manual inhibition data (3 replicates) were fit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065928#s2" target="_blank">Materials and Methods</a> section.</p

Binding properties of ketoconazole and ketaminazole with CaCYP51 and HsCYP51.

<p>Azole antifungals were progressively titrated against 5 µM CaCYP51 (filled circles) and 5 µM HsCYP51 (hollow circles). The resultant type II difference spectra are shown for ketoconazole (A) and ketaminazole (B). Saturation curves for ketoconazole (C) and ketaminazole (D) were constructed and a rearrangement of the Morrison equation (45) was used to fit the data. The data shown represent one replicate of the three performed.</p

Structures of LOX inhibitors.

<p>Structures of LOX inhibitors.</p