THE REACTION OF FLUOROCITRATE WITH ACONITASE AND THE CRYSTAL-STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX

It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of flu orocitrate is to inactivate aconitase, In this paper we present eviden ce supporting the hypothesis that the (-)-erythro diastereomer of 2-fl uorocitrate acts as a mechanism based inhibitor of aconitase bg first being converted to fluoro-cis-aconitate, followed by addition of hydro xide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn) , which binds very tightly, but not covalently, to the enzyme, Formati on of HTn by these reactions is in accord with the working model for t he enzyme mechanism, That HTn is the product of fluorocitrate inhibiti on is supported by the crystal structure of the enzyme-inhibitor compl ex at 2.05-Angstrom resolution, release of fluoride stoichiometric wit h total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC analysi s of the product, slow displacement of HTn by 10(6)-fold excess of iso citrate, and previously published Mossbauer experiments. When (+)-eryt hro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC analysis of the pr oducts indicates the formation of oxalosuccinate, and its derivative a lpha-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structu re of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural su bstrate, isocitrate. The structure exhibits four hydrogen bonds <2.7 A ngstrom in length involving HTn, H2O bound to the [4Fe-4S] cluster, As p-165 and His-167, as well as low temperature factors for these moieti es, consistent with the observed very tight binding of the inhibitor.