THE DIOXYGENATION RATE IN LIPOXYGENASE CATALYSIS IS DETERMINED BY THEAMOUNT OF IRON(III) LIPOXYGENASE IN SOLUTION

The dioxygenation rate in reactions catalyzed by lipoxygenase-1 from s oybeans has been measured as a function of the enzyme present in the F e(III) form with rapid kinetic techniques. The experiments were carrie d out at pH 10, 25-degrees-C. The product concentration and the fracti on of iron(III) lipoxygenase were monitored by measuring the absorbanc e at 243 nm and the tryptophan fluorescence at 3 30 nm (excitation at 287 nm), respectively. In reactions started with 1.3 muM iron(II) lipo xygenase and 9 muM linoleate, the initial rate, r(init) (estimated fro m the increase in absorbance over the initial 0.02 s of the reaction), is very small (4 s-1). In contrast, when the reactions are started wi th 1.3 muM iron(III) lipoxygenase, r(init) is large (150 s-1). In reac tions started with mixtures of iron(II) and iron(III) lipoxygenase, r( init) is linearly related to the initial concentration of the Fe(III) enzyme form. Redistributions of the Fe(II) and Fe(III) enzyme forms du ring the reaction with 12 nM enzyme and 10, 50, or 100 muM linoleate a ppear to be directly reflected in changes in the dioxygenation rate, T he observations provide solid evidence for the hypothesis that only ir on(III) lipoxygenase can catalyze the hydrogen abstraction step in the dioxygenation reaction, and thus can be regarded as the active enzyme species. The observed dynamics are accurately predicted by a nonallos teric, two-step model for lipoxygenase catalysis [Schilstra et al. (19 92) Biochemistry 31, 7692 7699].