The kinetic behaviour of a two-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 biphasic media (octane/buffer pH 9.6, v/v = 1:1). The lipase-catalysed hydrolysis of trilinolein and the subsequent lipoxygenation of the liberated linoleic acid, were coupled in a modified Lewis cell with a well-defined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipase at the organic/aqueous interface. Linoleic acid, liberated after hydrolysis was transferred to the aqueous phase and reacted with lipoxygenase. This reaction consumed linoleic acid and produced hydroperoxides, which favoured the transfer of residual linoleic acid, since they possess surface active properties. Catalysis and transfer influenced each other reciprocally. At low substrate concentrations, cooperativity phenomena were observed in the experimental and also the modelled two-enzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medium, seemed to be independent of the substrate concentration, unlike that observed in homogeneous monophasic enzymology. The numerical integration program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) trilinolein hydrolysis in biphasic medium, (2) linoleic acid transfer across a liquid/liquid interface and (3) lipoxygenation in an aqueous media. Results obtained by modelling were similar to the results observed experimentally.