title: Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability.
abstract: Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a approximately 30 kDa N-terminal domain and a approximately 60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a approximately 60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of alpha-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 x 10(6) and 10.9 x 10(6) M(-1) x s(-1), respectively), the activation energy of the reaction being 4.5 +/- 0.5 and 8.3 +/- 0.9 kJ x mol(-1) for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, alpha-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio- and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inhibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio(patho)logical conditions.
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, University of Rome, Tor Vergata, Rome, Italy. maccarone@med.uniroma2.it
authors.forename: M
authors.initials: M
authors.lastname: Maccarrone
authors.forename: M L
authors.initials: ML
authors.lastname: Salucci
authors.forename: G
authors.initials: G
authors.lastname: van Zadelhoff
authors.forename: F
authors.initials: F
authors.lastname: Malatesta
authors.forename: G
authors.initials: G
authors.lastname: Veldink
authors.forename: J F
authors.initials: JF
authors.lastname: Vliegenthart
authors.forename: A
authors.initials: A
authors.lastname: Finazzi-Agrò
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013025:Glycine max / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*
chemical_list: D008081:Liposomes; D008565:Membrane Proteins; D010446:Peptide Fragments; D008084:Lipoxygenase; D014357:Trypsin