The steady-state kinetics of the oxygenation of linoleic acid catalysed by soybean lipoxygenase-I were studied. The results showed that lipoxygenase-1 is strongly inhibited by its substrate, linoleic acid. In the presence of the product of the reaction, 13-~,-hydroperoxy-linoleic acid, the substrate inhibition only affects the apparent affinity for O2 and is of a hyperbolic type.A kinetic scheme of the oxygenation reaction is presented, which postulates two substrate-binding sites on the enzyme, one for linoleic acid and one for O,, and a regulatory binding site, which can either bind the product or the fatty acid substrate.Since previous studies indicated that the product of the reaction influences the oxidation state of the iron present in protein, the steady-state kinetics of the native enzyme and of the enzyme pre-incubated with the product were compared.Pre-incubation of the enzyme with the product did not lead to altered steady-state kinetics of the reaction compared to those of the native enzyme.Lipoxygenase type I from soybeans, a mononuclear non-heme-iron(Fe")-containing dioxygenase, catalyzes the conversion of linoleic acid and several other poly-unsaturated fatty acids with a 1,4-cis,cis-pentadiene structure [l] into (n-6)h-hydroperoxy fatty acids. Smith and Lands [2] studied the steady-state kinetics of the formation of the hydroperoxides from several fatty acid substrates in air-saturated solutions.They demonstrated that the enzyme is activated by the product hydroperoxide. Since the reaction is inhibited by trapping of hydroperoxide formed, they proposed, that activation by the product is compulsory.Garssen et al. [3] demonstrated that under anaerobic conditions both the fatty acid substrate (linoleic acid) and the (n-6)~,-hydroperoxide are converted by the enzyme into secondary products, among which are fatty acid dimers, oxodienoic acids and n-pentane. Electron paramagnetic resonance (EPR) studies by De Groot et al. [4] showed that this anaerobic reacAbbreviations. R-13-00H, 13-~,-hydroperoxy-linoleic acid;Enzyme. Lipoxygenase or linoleate: oxygen oxidoreductase (EC EPR, electron paramagnetic resonance.1.13.1 1.12).tion is a radical process, involving linoleic acid freeradicals created by the enzyme. The anaerobic reaction only takes place in the presence of the (n-6)~,-hydroperoxide. Recently it was found that 13-~,-hydroperoxy-linoleic acid (R-13-00H) affects the fluorescence [5], absorbance [6] and EPR spectra [7] of lipoxygenase-1. These studies revealed that an equimolar amount of R-I 3-00H converts the native enzyme from the ferrous state (EPR silent; no absorption maxima in the visible region) into the ferric state (EPR signals aroundg = 6; absorption maximum at about 330nm). Furthermore a molar excess of R-13-00H over enzyme gives rise to the appearance of a second absorption band (at 580 nm) and a strong EPR signal at g=4.3, whereas the signals around g = 6 are reduced in intensity [8]. When linoleic acid is added to the enzyme in the ferric state under anaerobic conditions, the enzyme ...
