Kinetic study of lipoxygenase-hydroperoxylinoleic acid interaction

Interaction of lipoxygenase with hydroperoxylinoleic acid, which is the product of this enzyme reaction and acts as an activator, was studied kinetically by the fluorescence stopped-flow method. The kinetic features are consistent with a two-step mechanism involving a fast bimolecular association process followed by a slow unimolecular process. The dissociation constant of the bimolecular process was 3 (+/-2) - 10(-5) M, which was appreciably dependent on temperature and pH, in contrast to the rate constant of the latter process. The enthalpy and the entropy of activation for the unimolecular process were estimated to be 21 kcal/mol and 20 e.u., respectively. The pH dependence of the rate constant indicated that an ionizable group with pK of about 8.6 is involved in the interaction. Linoleic acid, the substrate of lipoxygenase, and oleic acid inhibited the interaction between the lipoxygenase and the hydroperoxylinoleic acid by reducing the rate. A series of saturated monohydric alcohols also reduced the rate of the interaction as the chain length of the alcohols increases, though methanol and ethanol increased the rate of the interaction.