Effect of lipid hydroperoxide on lipoxygenase kinetics

M. Schilstra, G.A. Veldink, J. Verhagen, J.F.G. Vliegenthart

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    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, 25 OC, and 240 pM 02 with rapid kinetic techniques. The following observations were made: (1) Iron(I1) and iron(II1) lipoxygenases are kinetically different: reactions started with the Fe(I1) enzyme form show a lag phase, whereas iron(II1) lipoxygenase induces an initial burst. (2) Oxidation of the enzyme alone is not sufficient to abolish the lag phase: at [SI > 50 pM, the initial burst in the iron(II1) lipoxygenase curves is still followed by a lag. The lag phase disappears completely only in the presence of micromolar quantities of P. (3) The approximate dissociation constants for S and P are 15 and 24 pM, respectively, 1 order of magnitude smaller than the corresponding values in the absence of oxygen. The observed kinetics are predicted by numerical integration of the rate equations of a model based on the single lipid binding site mechanism for the anaerobic lipoxygenase reaction [Ludwig et al. (1987) Eur. J. Biochem. 168,325- 337; Verhagen et al. (1978) Biochim. Biophys. Acta 529, 369-3791, A quasi-steady-state approximation of the model suggests that at high [S]/[P] the fraction of active iron(II1) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.
    Original languageEnglish
    Pages (from-to)7692-7699
    JournalBiochemistry
    Volume31
    Issue number33
    DOIs
    Publication statusPublished - 1992

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