Molecular Pharmacology, Vol 10, 130-139, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Inhibition of Liver Aconitase Isozymes by (-)-erythro-Fluorocitrate

¹ Departments of Biochemistry and Biophysics, Pharmacology, and Cardiovascular Research Institute, University of California, San Francisco, California 94143

A highly purified cytosol aconitase [citrate (isocitrate) hydrolyase, EC 4.2.1.3] was isolated from pig liver. Whereas purified aconitase preparations previously reported in the literature require cysteine and Fe²⁺ for maximal catalytic activity, the purified cytosol enzyme and the partially purified mitochondrial aconitase described here need no added activator. The molecular weight of the purified cytosol enzyme as determined by sucrose density gradient centrifugation was between 107,000 and 111,000. Inhibitory effects of (-)-erythro-fluorocitrate on both aconitase isoenzymes were studied in the absence and presence of Mg²⁺ and Mn²⁺. Fluorocitrate was a competitive reversible inhibitor of both aconitases, as deduced from steady-state kinetic analyses (average K_i = 22-45 µM). The bivalent cations Mg²⁺ and especially Mn²⁺, when incubated with either aconitase isoenzyme, inhibited enzymatic activity, an effect which increased with time. A high concentration (30 mM) of citrate reversed the inhibition and protected the enzyme against inhibition by Mg²⁺ or Mn²⁺. When (-)-erythro-fluorocitrate and Mg²⁺ (or Mn²⁺) were present simultaneously, as in the isocitrate dehydrogenase coupled assay system, the time-dependent inhibition which ensued was a result of competitive inhibition by fluorocitrate and the more complex inhibition of the enzyme by Mg²⁺ or Mn²⁺

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ACKNOWLEDGMENT It is a pleasure to acknowledge the contribution of Miss Barbara Lancaster to the writing of the manuscript.

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