Enzyme-enzyme interactions as modulators of the metabolic flux through the citric acid cycle

A general analysis of the regulation of the citric acid cycle is hampered by the intimate interplay believed to exist between the various surrounding pathways. Two main regulatory mechanisms are thought to determine the flux through the cycle: (1) regulation of individual cycle enzymes, and (2) reversible complex formation between various enzymes of the cycle and related pathways. The latter mechanism allows a cell to maintain a high flux of substrates with a moderate number of intermediates, and offers a means of metabolite channeling. We were able to demonstrate specific interactions between several vertebrate cycle enzymes in conditions of reduced water concentration, i.e. by using immobilized enzyme systems. From affinity chromatographic experiments, we have shown that the enzymes of the citric acid cycle and the aspartate-malate shuttle are organized as one huge multi-enzyme complex, and a stoichiometric arrangement of fumarase/malate dehydrogenase/citrate synthase/aspartate aminotransferase has been postulated. Affinity electrophoresis was used as a new experimental device by which the enzyme-enzyme interactions could be directly visualized.