Effect of lipid hydroperoxide on lipoxygenase kinetics
1992
Schilstra, M.J. | Veldink, G.A. | Verhagen, J. | Vliegenthart, J.F.G.
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 micromolar) on linoleic acid (S, 1-80 micromolar) oxygenation catalysis by 12 nM lipoxygenase-1 from soybean was studied at pH 10, 25 degrees C, and 240 micromolar O2 with rapid kinetic techniques. The following observations were made: (1) Iron(II) and iron(III) lipoxygenases are kinetically different: reactions started with the FE(II) enzyme form show a lag phase, whereas iron(III) lipoxygenase induces an initial burst. (2) Oxidation of the enzyme alone is not sufficient to abolish the lag phase: at [S] > 50 micromolar, the initial burst in the iron(III) 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 micromolar, 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, 369-379]. A quasi-steady-state approximation of the model suggests that at high [S]/[P] the fraction of active iron(III) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.
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