title: Kinetic and structural investigations into the allosteric and pH effect on the substrate specificity of human epithelial 15-lipoxygenase-2.
abstract: 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-(S)-hydroxy-(9Z,11E)-octadecadienoic acid [13-(S)-HODE] and 13-(S)-hydroperoxy-(6Z,9Z,11E)-octadecatrienoic acid to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the kcat/KM substrate specificity ratio of arachidonic acid (AA) and γ-linolenic acid (GLA) by 4-fold. 13-(S)-HODE achieves this change by activating kcat/KM(AA) but inhibiting kcat/KM(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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Netra
authors.initials: N
authors.lastname: Joshi
authors.forename: Eric K
authors.initials: EK
authors.lastname: Hoobler
authors.forename: Steven
authors.initials: S
authors.lastname: Perry
authors.forename: Giovanni
authors.initials: G
authors.lastname: Diaz
authors.forename: Brian
authors.initials: B
authors.lastname: Fox
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
mesh_terms: D000495:Allosteric Site; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity
chemical_list: C458528:ALOX15B protein, human; D001093:Arachidonate 15-Lipoxygenase