title: Structural basis for pH-dependent alterations of reaction specificity of vertebrate lipoxygenase isoforms.
abstract: Lipoxygenases have been classified according to their specificity of fatty acid oxygenation and for several plant enzymes pH-dependent alterations in the product patterns have been reported. Assuming that the biological role of mammalian lipoxygenases is based on the formation of specific reaction products, pH-dependent alterations would impact enzymes' functionality. In this study we systematically investigated the pH-dependence of vertebrate lipoxygenases and observed a remarkable stability of the product pattern in the near physiological range for the wild-type enzyme species. Site-directed mutagenesis of selected amino acids and alterations in the substrate concentrations induced a more pronounced pH-dependence of the reaction specificity. For instance, for the V603H mutant of the human 15-lipoxygenase-2 8-lipoxygenation was dominant at acidic pH (65%) whereas 15-H(p)ETE was the major oxygenation product at pH 8. Similarly, the product pattern of the wild-type mouse 8-lipoxygenase was hardly altered in the near physiological pH range but H604F exchange induced strong pH-dependent alterations in the positional specificity. Taken together, our data suggest that the reaction specificities of wild-type vertebrate lipoxygenase isoforms are largely resistant towards pH alterations. However, we found that changes in the assay conditions (low substrate concentration) and introduction/removal of a critical histidine at the active site impact the pH-dependence of reaction specificity for some lipoxygenase isoforms.
authors.affiliation: Institute of Biochemistry, University Medicine Berlin - Charité, Germany. matthias.walther@charite.de
authors.forename: M
authors.initials: M
authors.lastname: Walther
authors.forename: J
authors.initials: J
authors.lastname: Roffeis
authors.forename: C
authors.initials: C
authors.lastname: Jansen
authors.forename: M
authors.initials: M
authors.lastname: Anton
authors.forename: I
authors.initials: I
authors.lastname: Ivanov
authors.forename: H
authors.initials: H
authors.lastname: Kuhn
mesh_terms: D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D051379:Mice; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D050505:Mutant Proteins; D001091:Arachidonate Lipoxygenases; C515716:ALOX15 protein, human; D001093:Arachidonate 15-Lipoxygenase; C054192:arachidonate 8-lipoxygenase; D010100:Oxygen