Effect of lipid hydroperoxide on lipoxygenase kinetics
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.