Fluorocitrate inhibition of aconitase. Reversibility of the inactivation

Abstract

Fluoride ion is released nearly stoichiometrically when (−)- erythro-fluorocitrate is incubated with aconitase. The release of F − parallels the loss in activity and could arise from direct displacement of F − by a base on the enzyme or from dehydration to fluoro- cis-aconitate and attack of an enzymic base to release F −. Aconitase inactivated by 14C-fluorocitrate does not retain radioactivity when passed through G-50 Sephadex or precipitated by ammonium sulfate. Full enzymic activity can be regained after either of these treatments by activation by cysteine and ferrous salts. These data are consistent with the report of fluorocitrate being a competitive (and non-competitive) inhibitor of aconitase (Villafranca, J.J. (1972) Intra-Science Chem. Rept. 6 (4), 1–11) which rapidly inactivates the enzyme. This inactivated enzyme may be a very labile covalent complex, a very tight complex between enzyme and fluoro- cis-aconitate or a tight complex between a defluorinated deravitive of fluorocitrate. In the course of Peters (1957) extensive work on the toxic effects of fluoroacetate, he determined that fluoroacetate was metabolically converted to fluorocitrate. This finding and the fact that citrate levels rise soon after ingestion of fluoroacetate led to the suggestion that fluorocitrate inactivates aconitase (E.C. 4.2.1.2). Recently, conflicting reports concerning the site of inactivation in mitochondria by the inhibiting isomer of fluorocitrate, (−)- erythro-fluorocitrate (1R:2R, 1-fluoro-2-hydroxy-1,2,3-propanetricarboxylate) have appeared (Eanes et al. 1972; Brand et al, 1973). Eanes et al. (1972) contends that the tricarboxylate carrier is the site of inhibition, while Brand et al. (1973) has compelling evidence that aconitase is the site of inhibition. This controversy is a matter of intrepretation of the results and a greater knowledge of the inactivation of aconitase by fluorocitrate may be useful in these interpretations. The results reported herein are concerned with the mechanism of inactivation of purified mitochondrial aconitase by fluorocitrate and demonstrate that this reaction is readily reversible.