Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes.
To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004058:Diffusion; D004800:Enzymes, Immobilized; D066298:In Vitro Techniques; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008956:Models, Chemical; D009206:Myocardium / Q000201:enzymology; D012996:Solutions; D013552:Swine
D004800:Enzymes, Immobilized; D012996:Solutions; D008291:Malate Dehydrogenase; D001219:Aspartate Aminotransferases