Factors affecting the initial rate of lipoxygenase catalysis

The rate of peroxidation of linoleic acid by soybean type-1 lipoxygenase was studied under conditions which assured that the substrate was present as a monomolecular solution and that the first 5% of the reaction was observed. In order to achieve this, the kinetics were carried out at pH 10.0 in borate buffer using linoleic acid and enzyme concentrations of less than 75 μM and 0.2 nM respectively. The initial rate was increased by the presence of added product (13-hydroperoxy-9(Z),11(E)-octadecadienoic acid) in the substrate solutions in a concentration dependent and saturatable fashion. Product analogues lacking the hydroperoxide group (13-hydroxy-9(Z),11(E)-octadecadienoic acid and 13-methoxy-9(Z),11(E)-octadecadienoic acid) did not evoke this rate enhancing effect. These compounds reduced the initial rate when preincubated with enzyme prior to mixing with substrate. The results indicated that the chemical reactivity of the product was a necessary requirement for its activating effect on the enzyme.