ABSTRACT:In order to investigate the activation of lipoxygenase and to clarify the role of the oxygenation product hydroperoxide in this process, the effect of 13-hydroperoxylinoleic acid (P, 0-35 pM) on linoleic acid (S, 1-80 pM) oxygenation catalysis by 12 nM lipoxygenase-1 from soybean was studied at pH 10, (II1) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.

The kinetic parameters of the first and second oxygenation of arachidonic acid by soybean lipoxygenase-1 were determined and found to be for the first step at pH 10.0, Km (arachidonic acid) = 8.5 +/- 0.5 microM; kcat = 225 +/- 7 s-1 and for the second step at pH 8.7 Km (15-HPETE) = 440 +/- microM; kcat = 25 +/- 1 s-1. In the second oxygenation for which 15-Ls-hydroperoxy 5-cis, 8-cis, 11-cis 13-trans-eicosatetraenoic acid is a substrate, two isomeric dihydroperoxy fatty acids are formed. After separation of the corresponding dihydroxy esters by high-performance liquid chromatography, they were identified by mass-spectrometry, 1H- and 13C-NMR spectroscopy as 8-DS, 15-LS-dihydroperoxy 5-cis, 9-trans, 11-cis, 13-trans-eicosatetraenoic acid and 5-DS, 15-LS-dihydroperoxy 6-trans, 8-cis, 11-cis, 13-trans-eicosatetraenoic acid. Independent evidence for the absolute configurations was obtained by capillary gas-liquid chromatography of diastereomeric R-(-)-2-butyl esters of the acetylated 2-hydroxy carboxylic acids produced by oxidative ozonolysis of the acetylated dihydroxy fatty acids. It is concluded that soybean lipoxygenase-1 produces hydroperoxides with predominantly the S-configuration irrespective of the position in the fatty acid which is oxygenated.

The steady-state kinetics of the anaerobic reaction of soybean lipoxygenase-1 with linoleic acid and 13-L-hydroperoxylinoleic acid were studied. Initial rates of the formation of oxodienoic acids**, absorbing at 285 nm, were measured at pH 10. About 50% of the consumed 13-L-hydroperoxylinoleic acid was converted into oxodienoic acids regardless of the initial ratio of the two substrates. A linear inhibition by both linoleic acid and 13-L-hydroperoxylinoleic acid was observed in the concentration range studied, which is on the upper side limited by the concentrations at which micelle- or acid-soap formation starts. A kinetic scheme is proposed based on one active site in lipoxygenase-1 which alternately binds the two substrates. Values for the kinetic constants were calculated by fitting simultaneously the complete set of data to the appropriate rate equation.

No abstract

Surface tension measurements of linoleic acid solutions in 0.1 M sodiumborate buffer pH 10 at 23 degrees C showed that at increasing the linoleic acid concentration a sharp transition from monomers to micelles occurs at 167 micrometer. At pH 9 and 8 formation of acid-soap dimers from monomers starts at 60 micrometer and 21 micrometer respectively. The concentration range at which only monomers exist is therefore markedly reduced. For 13-L-hydroperoxylinoleic acid at pH 10 acid-soap formation still takes place, starting at approx. 220 micrometer. The total lipid concentration at which acid-soap or micelle formation starts in mixtures of linoleic acid and 13-L-hydroperoxylinoleic acid has been determined in relation to the molar ratio of both acids.