THE KINETIC-BEHAVIOR OF A 2-ENZYME SYSTEM IN BIPHASIC MEDIA - COUPLING HYDROLYSIS AND LIPOXYGENATION

Analysis of the kinetic behaviour of a two-enzyme-system carrying out two consecutive reactions was investigated in macroheterogeneous bipha sic media (octane/buffer pH 9.6, v/v = 1:1). The lipase-catalysed hydr olysis of trilinolein and the subsequent lipoxygenation of the liberat ed linoleic acid, were coupled in a modified Lewis cell with a well-de fined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipas e at the organic/aqueous interface. Linoleic acid, liberated after hyd rolysis was transferred to the aqueous phase and reacted with lipoxyge nase. This reaction consumed linoleic acid and produced hydroperoxides , which favoured the transfer of residual linoleic acid, since they po ssess surface active properties. Catalysis and transfer influenced eac h other reciprocally. At low substrate concentrations, cooperativity p henomena were observed in the experimental and also the modelled two-e nzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medi um, seemed to be independent of the substrate concentration, unlike th at observed in homogeneous monophasic enzymology. The numerical integr ation program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) tr ilinolein hydrolysis in biphasic medium, (2) linoleic acid transfer ac ross a liquid/liquid interface and (3) lipoxygenation in an aqueous me dia. Results obtained by modelling were similar to the results observe d experimentally.