title: Inhibitory and mechanistic investigations of oxo-lipids with human lipoxygenase isozymes.
abstract: Oxo-lipids, a large family of oxidized human lipoxygenase (hLOX) products, are of increasing interest to researchers due to their involvement in different inflammatory responses in the cell. Oxo-lipids are unique because they contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and thus increase inhibitor potency. Due to the resemblance of oxo-lipids to LOX substrates, the inhibitor potency of 4 different oxo-lipids; 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE), 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-ETE), and 13-oxo-9,11-(Z,E)-octadecadienoic acid (13-oxo-ODE) were determined against a library of LOX isozymes; leukocyte 5-lipoxygenase (h5-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), human platelet 12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2 (h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit reticulocyte 15-LOX (r15-LOX). 15-Oxo-ETE exhibited the highest potency against h12-LOX, with an IC₅₀=1 ± 0.1 μM and was highly selective. Steady state inhibition kinetic experiments determined 15-oxo-ETE to be a mixed inhibitor against h12-LOX, with a Kic value of 0.087 ± 0.008 μM and a Kiu value of 2.10 ± 0.8 μM. Time-dependent studies demonstrated irreversible inhibition with 12-oxo-ETE and h15-LOX-1, however, the concentration of 12-oxo-ETE required (Ki=36.8 ± 13.2 μM) and the time frame (k₂=0.0019 ± 0.00032 s(-1)) were not biologically relevant. These data are the first observations that oxo-lipids can inhibit LOX isozymes and may be another mechanism in which LOX products regulate LOX activity.
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064, United States.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Armstrong
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064, United States.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Diaz
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064, United States.
authors.forename: Victor
authors.initials: V
authors.lastname: Kenyon
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064, United States. Electronic address: holman@ucsc.edu.
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D016859:Lipoxygenase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D058945:Lipoxygenases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013025:Glycine max / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001095:Arachidonic Acids; D007527:Isoenzymes; D016859:Lipoxygenase Inhibitors; D058945:Lipoxygenases
keywords: Inhibitor; Lipoxygenase; Mechanism; Oxo-lipids