CUCUMBER COTYLEDON LIPOXYGENASE OXYGENIZES TRILINOLEIN AT THE LIPID WATER INTERFACE/

The reactivity of cucumber cotyledon lipoxygenase with trilinolein was examined. The activity of the enzyme against linoleic acid rapidly de creased with increasing pH of the assay solution, and essentially no a ctivity could be detected above pH 8.5. The rapid decrease in activity was not the result of an inactiveness of the enzyme at alkaline pH, b ecause with trilinolein, the enzyme showed a broad pH-activity profile , and substantial activity could be detected even at pH 9.0. Rather, t he decrease in activity was due to the dissociation of the linoleic ac id emulsion into acid-soap aggregates and/or the monomeric form, depen ding on the ionization of the terminal carboxylic group. This suggests that cucumber cotyledon lipoxygenase acts only on an insoluble substr ate at the lipid/water interface but not on a soluble one. High-perfor mance liquid chromatography analyses of the products formed from trili nolein revealed that the enzyme inserted oxygen into the acyl moiety o f trilinolein without hydrolysis of the ester bonds. Preincubation of the enzyme with triolein emulsions effectively abolished its activity against trilinolein added afterward. Furthermore, the enzyme was adsor bed on the trilinolein or triolein emulsion droplets in an essentially irreversible manner. A reaction velocity curve of the enzyme with tri linolein showed saturation kinetics. This is thought to be due to a re gional substrate deficiency as the reaction proceeds. These lines of e vidence indicate that the enzyme, once bound to the lipid/water interf ace, is unable to break free and bind to other emulsions.