TY - JOUR
T1 - Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDA fragment with improved activity and membrane binding ability
AU - Maccarrone, M.
AU - Salucci, M. L.
AU - Van Zadelhoff, G.
AU - Malatesta, F.
AU - Veldink, G.
AU - Vliegenthart, J. F G
AU - Finazzi-Agrò, A.
PY - 2001/6/12
Y1 - 2001/6/12
N2 - 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 α-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 × 106 and 10.9 × 106 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, α-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.
AB - 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 α-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 × 106 and 10.9 × 106 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, α-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.
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U2 - 10.1021/bi010187m
DO - 10.1021/bi010187m
M3 - Article
C2 - 11389595
AN - SCOPUS:0035849587
VL - 40
SP - 6819
EP - 6827
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 23
ER -