Aconitase activated with Fe(2+), cysteine and ascorbate incorporates 1 g-atom of Fe(2+)/mol. Loss of this Fe(2+) by transfer to ferrozine, a Fe(2+) chelator, results in loss of activity. Ascorbate increases the rate of transfer of the essential Fe(2+) whereas citrate retards the rate of transfer. Transfer of Fe(2+) from inactive aconitase, 2 g-atoms of Fe/mol, can be accomplished in the presence of urea and ascorbate. The correlation of activity with the presence of an added g-atom of Fe(2+)/mol leads to the conclusion that active aconitase has only one active site per mol.
Methyl-m-aconitate behaves as a typical substrate for aconitase, two hydroxy acids, a-methylisocitric acid and -methylcitric acid, resulting from the enzyme-catalyzed hydration. Enzyme-catalyzed hydration to yield -methylisocitric acid is trans and, presumably, optical configurations of the two a-methylhydroxy acids are identical with those of D-z/zz-ey-isocitric acid and citric acid.Kn at pH 7.5, 28°, is 6.0 X 10~4 m and rela-A A Xconitase (EC 4.2.1.3 m-aconitase, aconitate hydratase, citrate (isocitrate) hydro-lyase) catalyzes by reversible hydration the equilibration of czs-aconitic acid, D-r/zz-eo-isocitric acid, and citric acid. The stereochemical configurations of the components of the system are such that, as depicted in Figure 1, frontal addition of H and OH to the a carbon of m-aconitic acid and addition from the rear of OH and H to the ß carbon is required for the equilibration (Gawron et al., 1961;Hanson and Rose, 1963
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