KINETIC-ANALYSIS OF THE INDUCTION PERIOD IN LIPOXYGENASE CATALYSIS

The dioxygenation of 50 muM linoleate at 0.1 muM (13S)-hydroperoxylino leate, 240 muM O2, pH 10, and 25-degrees-C, catalyzed by varying amoun ts of soybean lipoxygenase-1, was studied with rapid kinetic technique s. The aim was to assess the effect of transient redistributions of th e Fe(II) and Fe(III) enzyme forms on the shape of the reaction progres s curves. Reactions initiated with iron(II) lipoxygenase show an initi al increase in rate, the ''kinetic lag phase'' or ''induction period'' . The duration of this induction period varies from approximately 1 s at [lipoxygenase] > 20 nM to 5 s at [lipoxygenase] = 3 nM. At [lipoxyg enase] < 2 nM, the duration of the induction period in these curves is inversely proportional to [lipoxygenase]. The integrated steady-state rate equation for the single fatty acid binding site model of lipoxyg enase catalysis [Schilstra et al. (1992) Biochemistry 31, 7692-7699] a lso shows an induction period whose duration is inversely proportional to [lipoxygenase]. These observations, in combination with non-steady -state numerical simulations, lead to the conclusion that, at [lipoxyg enase] < 2 nM, pre-steady-state redistributions of enzyme intermediate s occur fast with respect to the rate at which the concentrations of s ubstrates and products change. At higher lipoxygenase concentrations, the pre-steady-state redistributions contribute significantly to the i nduction period. From a nonlinear least-squares fit to the steady-stat e rate equation of data obtained at lipoxygenase concentrations of 0.5 and 1 nM, it was calculated that 1% of the linoleate radicals that ar e formed after hydrogen abstraction dissociate from the active site be fore enzymic oxygen insertion has occurred.