title: Effect of lipid hydroperoxide on lipoxygenase kinetics.
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 microM) on linoleic acid (S, 1-80 microM) oxygenation catalysis by 12 nM lipoxygenase-1 from soybean was studied at pH 10, 25 degrees C, and 240 microM 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] greater than 50 microM, 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 microM, 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 a high [S]/[P] the fraction of active iron(III) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.
authors.affiliation: Department of Bioorganic Chemistry, University of Utrecht, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Schilstra
authors.forename: G A
authors.initials: GA
authors.lastname: Veldink
authors.forename: J
authors.initials: J
authors.lastname: Verhagen
authors.forename: J F
authors.initials: JF
authors.lastname: Vliegenthart
mesh_terms: D007700:Kinetics; D008041:Linoleic Acids / Q000138:chemical synthesis / Q000494:pharmacology*; D008054:Lipid Peroxides / Q000494:pharmacology*; D008084:Lipoxygenase / Q000378:metabolism*; D008433:Mathematics; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D013025:Glycine max / Q000201:enzymology
chemical_list: D008041:Linoleic Acids; D008054:Lipid Peroxides; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D008084:Lipoxygenase