A novel, definitive test for substrate channeling illustrated with the aspartate aminotransferase malate dehydrogenase system

A novel method is presented that establishes definitively the existence or nonexistence of direct metabolite transfer between consecutive enzymes in a metabolic sequence. The procedure is developed with the specific example o f channeling of oxaloacetate: between Escherichia coli aspartate aminotrans ferase (AATase) and malate dehydrogenase (MDH). The assay is carried out in the presence of a large excess of inactive variants of AATase. These mutan ts would outcompete the much smaller quantities of wildtype AATase for any docking sites on MDH and thus decrease the rate of the coupled L-aspartate to oxaloacetale to malate sequence only if the direct metabolite transfer m echanism is operative. The results show that oxaloacetate is not transferre d directly from AATase to MDH because no decrease in rate was observed in t he presence of similar to 100 mu M inactive mutants. This concentration is 10 times the physiological AATase concentration, which was determined in th is work. The methodology can be applied generally.