On the mechanism of aconitase.

Analysis of aconitase kinetics shows that all three substrates, citrate, d(+)-isocitrate, and cis aconitate, are equivalent in the number of slow, rate determining steps in their inter-conversion. Kinetics and the inhibition studies using deuterium oxide indicate that cis aconitate is not an obligatory intermediate in the citrate: isocitrate conversion. The lack of deuterium incorporation from the medium into enzymatically formed citrate from isocitrate shows that conversion to involve and intramolecular hydrogen rearrangement and eliminates cis aconitate as the intermediate. The proposed mechanism is similar to known intramolecular hydrogen rearrangements, the Whitmore Shift of carbonium ions; It is though that the enzyme can induce carbonium ion formation in the substrate with the aid of ferrous iron. It is postulated that the metal serves as an electron acceptor or Lewis acid, and that the carbonium ion is formed as a result of coordination of the hydroxyl with the metal. The postulated mechanism accounts for all the reactions of he enzyme and for its kinetic behavior. Aconitase has been prepared from mitochondria by freezing and thawing. The soluble aconitase is more active than the mitochondrial enzyme in the isocitrate-aconitate and citrate-aconitate reactions, but mitochondrial aconitase is more active in the isocitrate-citrate transformation. Soluble aconitase showed an optimum at pH 7.3 but mitochondrial aconitase showed optima at pH 5.8 and 7.3. The isocitrate-aconitate and citrate-aconitate reactions of both soluble and mitochondrial aconitase were inhibited by o-phenanthroline and reactivated by ferrous ions plus ascorbate. Mitochondria incubated with isocitrate retain aconitate. The data on the intracellular distribution of aconitase were markedly affected by the condition of the assay.