Kinetic characterization of enhanced lipase activity on oil bodies.
Enzyme access, kinetic behavior, and protein-protein interactions are critical for explaining reaction of the metabolites contained within the myriad compartments of biological systems. To explore these relationships, the reaction kinetics of oil bodies versus oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the interface resulted in an enzyme-membrane complex that was catalytically enhanced 3-15-fold over the emulsion system for substrate concentrations in the measured range of approximately 1-5.5 mM. This disparity is explained by the availability of substrate to the enzyme active site (defined as the availability parameter "A") which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with Km replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/ mg protein. The interaction is verified through separation of the enzyme-membrane complex which shows nearly double the activity towards an emulsified soybean oil substrate (activity ratio of 5:3) when compared to the native enzyme.
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Biochemical Engineering Laboratory, Agricultural and Biological Engineering, Purdue University, F.S. Bldg Rm 3239, 745 Agricultural Mall Drive, West Lafayette, IN 47907-2009, USA.
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D003198:Computer Simulation; D004655:Emulsions / Q000737:chemistry*; D004789:Enzyme Activation; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry*; D008956:Models, Chemical; D013024:Soybean Oil / Q000737:chemistry*
D004655:Emulsions; D013024:Soybean Oil; D008049:Lipase