The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex

Abstract

It has been known for many years that f luoroacetate and f luorocitrate when metabolized are highly toxic, and that at least one effect of f luorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (؊)-erythro diastereomer of 2-f luorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to f luoro-cis-aconitate, followed by addition of hydroxide and with loss of f luoride 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 f luorocitrate inhibition is supported by the crystal structure of the enzymeinhibitor complex at 2.05-Å resolution, release of f luoride stoichiometric with total enzyme when (؊)-erythro-2-f luorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10 6 -fold excess of isocitrate, and previously published Mössbauer experiments. When (؉)-erythro-2-f luorocitrate is added to aconitase, the release of f luoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative ␣-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (؊)-erythro-2-f luorocitrate. 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 <2.7 Å in length involving HTn, H 2 O bound to the [4Fe-4S] cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.The mechanism of the inhibitory effects of fluorocitrate on the enzyme aconitase [citrate(isocitrate)hydrolyase, EC 4.2.1.3] has been a long-standing problem in biochemistry. The toxic nature of fluoroacetate was discovered over 50 years ago (1, 2) and citrate was found to accumulate in tissues poisoned with compounds that could provide the fluoroacetyl residue. On this basis Peters (3) and Martius (4) proposed that the inhibitory substance was a fluorotricarboxylic acid. Subsequently it was shown that 2-fluorocitrate is indeed produced metabolically via the citrate synthase reaction (5) and that in the presence of this substance the enzyme aconitase is inhibited (6). Aconitase catalyzes the conversion of citrate to isocitrate (Iso) via the obligatory intermediate cis-aconitate (Scheme I).

Staphylococcus epidermidis Polysaccharide Intercellular Adhesin Production Significantly Increases during Tricarboxylic Acid Cycle Stress

“…Fluorocitrate is a highly toxic compound when metabolized to 4-hydroxy-transaconitate by the TCA cycle enzyme aconitase (19,25). The toxicity is due to very tight, noncovalent binding of 4-hydroxytrans-aconitate to aconitase (19). As the concentration of fluorocitrate in the culture medium was increased, the amount of PIA produced increased ( ϭ 0.91) (Fig.…”

“…To test this hypothesis further, we incubated S. epidermidis strain SE1457 with a TCA cycle specific inhibitor, fluorocitrate, and determined the amount of cell-associated PIA. Fluorocitrate is a highly toxic compound when metabolized to 4-hydroxy-transaconitate by the TCA cycle enzyme aconitase (19,25). The toxicity is due to very tight, noncovalent binding of 4-hydroxytrans-aconitate to aconitase (19).…”

Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

“…Like acetate, FAc is converted into fluoroacetyl-CoA (7), which condenses with oxaloacetate (OAA) to produce (Ϫ)-erythro-(2R,3R)-2-fluorocitrate (2-FC) catalyzed by citrate synthase (CS) (8). It is generally assumed that as (Ϫ)-erythro-2-FC is a strong inhibitor of aconitase (9,10), the metabolic flow of the OTCA cycle is blocked and contributes to the toxicity of FAc. Sirevåg and Ormerod (11) reported decades ago that CO 2 assimilation and carbon flux are affected upon addition of FAc during phototrophic growth of the GSB Chlorobium limicola.…”

2-18F-Fluoroacetate: A Useful Tool for Assessing Gliosis in the Central Nervous System?

“…Subsequent release of fluoride ion and hydroxylation form 4-hydroxy-transaconitate (HTA). HTA has been shown to be a potent inhibitor of aconitase, and the resulting impairment of TCA cycle-mediated oxidative metabolism is the direct mechanism of fluoroacetate toxicity (17). FAC, like acetate, is preferentially taken up by astrocytes relative to neurons (18), and it follows that 2-18 F-fluoroacetate ( 18 F-FAC) could be a potentially useful noninvasive indicator of glial cell metabolism.…”