title: Probing conformational changes in lipoxygenases upon membrane binding: fine-tuning by the active site inhibitor ETYA.
abstract: Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme-membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors.
authors.affiliation: Department of Experimental Medicine and Surgery, Tor Vergata University of Rome, Via Montpellier 1, 00133 Rome, Italy.
authors.forename: Almerinda
authors.initials: A
authors.lastname: Di Venere
authors.forename: Eleonora
authors.initials: E
authors.lastname: Nicolai
authors.forename: Igor
authors.initials: I
authors.lastname: Ivanov
authors.forename: Enrico
authors.initials: E
authors.lastname: Dainese
authors.forename: Susan
authors.initials: S
authors.lastname: Adel
authors.forename: B C
authors.initials: BC
authors.lastname: Angelucci
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kuhn
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
authors.forename: Giampiero
authors.initials: G
authors.lastname: Mei
mesh_terms: D015117:5,8,11,14-Eicosatetraynoic Acid / Q000737:chemistry*; D020134:Catalytic Domain; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008084:Lipoxygenase / Q000737:chemistry*; D016859:Lipoxygenase Inhibitors / Q000737:chemistry*; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000201:enzymology
chemical_list: D008051:Lipid Bilayers; D016859:Lipoxygenase Inhibitors; D015117:5,8,11,14-Eicosatetraynoic Acid; D008084:Lipoxygenase
keywords: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N1-pyrenesulfonyl; 5,8,11,14-eicosatetraynoic acid; 6-dodecanoyl-N,N-dimethyl-2-naphthylamine; 8-anilino-1-naphthalenesulfonic acid; ANS; ETP; ETYA; FCS; FRET; Fluorescence dynamics; GUV; High pressure; LOX; LUV; Lipoxygenases; Motional flexibility; Py-PE; electron transfer particles; fluorescence correlation spectroscopy; fluorescence energy transfer; giant unilamellar vesicle; large unilamellar vesicle; laurdan; lipoxygenase; rabbit reticulocyte 12/15 lipoxygenase; rr 12/15-LOX; sb 15-LOX; soybean lipoxygenase 1