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 ∼30 kDa N-terminal domain and a ∼60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a ∼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 R-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 × 10 6 and 10.9 × 10 6 M -1 ‚s -1 , respectively), the activation energy of the reaction being 4.5 ( 0.5 and 8.3 ( 0.9 kJ‚mol -1 for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, R-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 inihibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio-(patho)logical conditions.Lipoxygenases are a family of monomeric non-heme, nonsulfur iron dioxygenases which catalyze the conversion of unsaturated fatty acids into conjugated hydroperoxides. Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1). They have been also implicated in atherosclerosis (2), brain aging (3), HIV infection (4), and kidney disease (5, 6). In plants, lipoxygenases favor germination and participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress (7). Recently, they have been also shown to initiate the programmed death (apoptosis) of plant cells (8). Soybean lipoxygenase-1 (LOX-1) 1 is widely used as a prototype for studying the homologous family of lipoxygenases from tissues of different species, both in structural (9-13) and in kinetic (14-18) investigations. The amino acid sequence (19) and three-dimensional structure (9, 10) of LOX-1 have been determined. The overall 839 amino acid residues of LOX-1, with a molecular mass of 93 840 Da, show 2 domains. The first 146 N-terminal amino acid residues form an 8-stranded -barrel, and the remaining 693 residues of the C-terminal domain are organized into 23 helices and 8 -strands. Mammalian lipoxygenases do ...

Structural Stability of Soybean Lipoxygenase-1 in Solution as Probed by Small Angle X-ray Scattering

“…26 Under the same experimental conditions, we found here that ETYA did not affect the membranebinding ability of LOX-1 nor its dependence on calcium ions (Table 2), whereas the addition of glycerol (5%) increased the binding of LOX-1 to membranes significantly (up to 175% of the controls), without affecting the calcium-dependence of this process (Table 2).…”

“…The structural effects of ETYA and glycerol were correlated with their effects on catalytic efficiency and membrane-binding ability of LOX-1, two critical aspects of the biological activity of the enzyme. 1,26 All our data point towards the remarkable structural stability of soybean LOX-1, whose crystal structure is essentially preserved in solution under various conditions.…”

“…We further investigated the effect of ETYA or glycerol on the catalytic efficiency and membranebinding ability of LOX-1, two essential aspects of the biological activity of this 1,26 as well as other lipoxygenases. 32 ETYA or glycerol were used in the same conditions (i.e.…”

Closing the Gate to the Active Site

“…Membrane Binding Assays-Negatively charged, unsaturated phosphatidylcholine liposomes were prepared in phosphate-buffered saline using the liposome kit (Sigma) as reported (17). The size of the vesicles was ϳ2 m, as determined under the microscope by comparison with the size (10 Ϯ 0.5 m) of Mono Q beads (Amersham Biosciences).…”

“…The size of the vesicles was ϳ2 m, as determined under the microscope by comparison with the size (10 Ϯ 0.5 m) of Mono Q beads (Amersham Biosciences). The binding of FAAH to liposomes was determined essentially as reported (17). Preliminary experiments showed that incubation of liposome suspensions corresponding to 1 mol of phosphatidylcholine (50 l) with 100 pmol of FAAH (corresponding to 2 M) for 0 -60 min at 25°C yielded the highest amount of bound FAAH (i.e.…”