Mechanism of Aconitase Action

Five experiments that support this scheme are presented. 1. The conversion of 3-T-isocitrate to citrate at early times gives T-citrate with no loss of tritium. 2. When the conversion of 2-T-citrate to isocitrate is brought to completion by trapping the isocitrate with isocitrate dehydrogenase, and the oc-ketoglutarate formed with glutamate dehydrogenase, the amount of tritium found in the glutamate agrees with the prediction based on the relative initial rates of formation of isocitrate and cis-aconitate from citrate. 3. The transfer of deuterium from citrate to isocitrate in the absence of high concentrations of cis-aconitate is shown to be largely intramolecular. 4. At high concentrations of cis-aconitate some of the tritium of 2-T-citrate that is normally found in the water is diverted to isocitrate. 5. This conservation of tritium is due to an intermolecular transfer since in the presence of high cis-aconitate the tritiated 2-methyl hydroxy acids give rise to tritiated isocitrate. These data support a mechanism of base-catalyzed proton abstraction from hydroxy acid substrate. The conjugate acid group so formed on the enzyme is slow to dissociate and either is transferred to bound cis-aconitate before the latter dissociates, or, if the cis-aconitate is released, the conjugate acid group dissociates to regenerate the enzyme base. In the interconversion of the hydroxy acids there is little or no transfer of 180-labeled hydroxyl group.