The Reaction of Fluorocitrate with Aconitase and the Crystal Structure of the Enzyme-Inhibitor Complex

• Published in: Proceedings of the National Academy of Sciences - PNAS 1996-11, Vol.93 (24), p.13699-13703
• Main Authors: Lauble, H., Kennedy, M. C., Emptage, M. H., Beinert, H., Stout, C. D.
• Format: Article
• Language: English
• Subjects: Aconitate Hydratase - antagonists & inhibitors; Aconitate Hydratase - chemistry; Aconitate Hydratase - metabolism; Animals; Biochemistry; Biological Sciences; Cattle; Citrates; Citrates - metabolism; Citrates - pharmacology; Crystallography, X-Ray; Crystals; Electron density; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Enzymes; Fluorides; Hydrogen bonds; Hydroxyls; Iron-Sulfur Proteins - antagonists & inhibitors; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - metabolism; Isocitrates - metabolism; Isomers; Mitochondria - enzymology; Models, Molecular; Molecules; Protein Binding; Stereoisomerism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.24.13699

It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of fluorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (-)-erythro diastereomer of 2-fluorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to fluoro-cis-aconitate, followed by addition of hydroxide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of fluorocitrate inhibition is supported by the crystal structure of the enzyme-inhibitor complex at 2.05- angstrom resolution, release of fluoride stoichiometric with total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 106-fold excess of isocitrate, and previously published Mossbauer experiments. When (+)-erythro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative α -ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds