Aconitase Reaction Research Articles

First Evidence of a Protective Effect of Plant Bioactive Compounds against H2O2-Induced Aconitase Damage in Durum Wheat Mitochondria.

In order to contribute to the understanding of the antioxidant behavior of plant bioactive compounds with respect to specific subcellular targets, in this study, their capability to protect aconitase activity from oxidative-mediated dysfunction was evaluated for the first time in plant mitochondria. Interest was focused on the Krebs cycle enzyme catalyzing the citrate/isocitrate interconversion via cis-aconitate, as it possesses a [4Fe-4S]2+ cluster at the active site, making it an early and highly sensitive target of reactive oxygen species (ROS)-induced oxidative damage. In particular, the effect on the aconitase reaction of five natural phenols, including ferulic acid, apigenin, quercetin, resveratrol, and curcumin, as well as of the isothiocyanate sulforaphane, was investigated in highly purified mitochondria obtained from durum wheat (DWM). Interestingly, a short-term (10 min) DWM pre-treatment with all investigated compounds, applied at 150 µM (75 µM in the case of resveratrol), completely prevented aconitase damage induced by a 15 min exposure of mitochondria to 500 µM H2O2. Curcumin and quercetin were also found to completely recover DWM-aconitase activity when phytochemical treatment was performed after H2O2 damage. In addition, all tested phytochemicals (except ferulic) induced a significant increase of aconitase activity in undamaged mitochondria. On the contrary, a relevant protective and recovery effect of only quercetin treatment was observed in terms of the aconitase activity of a commercial purified mammalian isoform, which was used for comparison. Overall, the results obtained in this study may suggest a possible role of phytochemicals in preserving plant mitochondrial aconitase activity, as well as energy metabolism, against oxidative damage that may occur under environmental stress conditions. Further investigations are needed to elucidate the physiological role and the mechanism responsible for this short-term protective effect.

Revisiting prochirality

We propose a new model for prochirality that satisfies all known examples: the prochiral plane. This plane contains the prochiral carbon and defines two separate faces for chemical modification. We extend this to enzyme catalysis, replacing the “three point attachment” hypothesis and its variants. Once a prochiral substrate is fixed on an enzyme surface, the asymmetry of the enzyme provides reactants exclusively on one side of the prochiral plane, producing an enantiomerically pure chiral product. The aconitase reaction is detailed as an example, using molecular modeling and its known enzymatic mechanism. We show that the prochiral substrate for this enzyme is not citrate, but rather cis-aconitate. The number of interaction points of cis-aconitate is not relevant to prochirality, but rather to substrate specificity. A second detailed example is the enzyme fumarase; here the substrate fumarate has only two binding sites, but is nonetheless fixed onto the enzyme and has a defined prochiral plane. We also provide a literature survey of more prochiral substrates, all of which have sp2 hybridized carbon and contain a prochiral plane. An example of a prochiral unnatural substrate for sphingosine kinase 2, fingolimod, has an sp3 hybridized prochiral carbon and also contains a prochiral plane. Finally, we provide an intuitive example of a prochiral physical object, a coffee cup, interacting with one hand and lip.

The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

Isocitrate dehydrogenase 2 (IDH2) is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP+-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD+ accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction), which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall “anoxic” glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2—frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia—catalyze reductive carboxylation of 2-oxoglutarate and reduction to D-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations. D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factor α.

Biochemical characterisation of aconitase from Corynebacterium glutamicum

In this work, aconitase of the biotechnologically relevant microorganism Corynebacterium glutamicum was characterised. The specific activity of aconitase in extracts of glucose-grown cells was determined by four different assays. In three of them the formation or disappearance of cis-aconitate was measured, whereas in the fourth assay the aconitase reaction was coupled with isocitrate dehydrogenase. The highest activity was determined with cis-aconitate as substrate (0.433 ± 0.054 U mg −1) and the lowest one with the coupled assay and citrate as substrate (0.134 ± 0.026 U mg −1). Only the latter assay covers the complete aconitase reaction and thus gives the most relevant information on in vivo activity. For the determination of kinetic constants, aconitase was heterologously overproduced, purified, reactivated and biochemically characterised. Size exclusion chromatography indicated that the protein is monomeric. The enzyme showed Michaelis–Menten kinetics with K m values of 480 ± 200 μM for citrate, 552 ± 302 μM for isocitrate and 18.5 ± 3.4 μM for cis-aconitate. The highest V max was observed for the hydration of cis-aconitate with 40.6 U mg −1. Aconitase was active over a wide pH and temperature range with maximal activity between pH 7.5 and 7.75 and at about 50 °C.

Iron-independent Phosphorylation of Iron Regulatory Protein 2 Regulates Ferritin during the Cell Cycle

Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: Mechanisms and relative contributions to aconitase inactivation

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO –). High NO concentrations are required to inhibit resting aconitase. Brief NO exposures led to a reversible inhibition competitive with isocitrate ( K I = 35 μM). Subsequently, an irreversible inactivation (0.65 M − 1 s − 1 ) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M − 1 s − 1 ). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 × 10 5 M − 1 s − 1 ). Carbon dioxide enhanced ONOO –-dependent inactivation via reaction of CO 3 − with the [4Fe-4S] cluster (3 × 10 8 M − 1 s − 1 ). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O 2 − and NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O 2 − production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O 2 − production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO − and CO 3 −, which also mediate m-aconitase tyrosine nitration, a footprint of reactive NO-derived species.

IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711-->Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1(S711E) displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1(S711E) retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1(S711E) completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1(S711E) is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.

The phosphinomethylmalate isomerase gene pmi, encoding an aconitase-like enzyme, is involved in the synthesis of phosphinothricin tripeptide in Streptomyces viridochromogenes.

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197-222, in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.

Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27-fold to a specific activity of approximately 55 micromol x min(-1) x mg(-1), based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 14 min was determined, clearly defining it as a thermostable protein. The apparent K(m) values for the three substrates cis-aconitate, citrate and isocitrate were found as 108 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino-acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR-spectroscopy, the enzyme hosts an interconvertible [3Fe--4S] cluster.

Identification of the high-molecular-mass mitochondrial oxaloacetate keto-enol tautomerase as inactive aconitase

The homogeneous bovine heart mitochondrial high-molecular-mass oxaloacetate keto-enol tautomerase [(1988) Biochim. Biophys. Acta 936, 10–19] is shown to be an iron-sulfur protein as revealed by the enzyme spectral properties and direct chemical determination of non-heme iron and acid-labile sulfur. The protein is capable of catalysing the aconitase reaction after treatment with ferrous ions under anaerobic conditions. Treatment of the ‘activated’ protein with N-ethylmaleimide results in the simultaneous irreversible loss of the oxaloacetate keto-enol tautomerase and aconitase activities. The effects of some substrates and inhibitors on both activities show that the same catalytic site is involved in the oxaloacetate tautomerase and aconitase reactions. It is concluded that the protein previously described as a 80 kDa oxaloacetate keto-enol tautomerase is inactive aconitase.

Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy.

The active form of aconitase has a diamagnetic [4Fe-4S]2+ cluster. A specific iron ion (Fea, which is lost during inactivation) is the binding site for substrate, as shown by Mössbauer spectroscopy. We have studied the mode of substrate and analogue binding at equilibrium to the paramagnetic [4Fe-4S]+ cluster of the reduced active form by 17O and 13C electron-nuclear double resonance spectroscopy with specifically labeled substrates. The data show that with substrate, only the carboxyl at C-2 of the propane backbone is strongly bound in addition to H2O or OH- (HxO) from the solvent, whereas in an isocitrate analogue that has a nitro group at C-2, the carboxyl and hydroxyl at C-1 are bound along with solvent HxO. We conclude from these data that, on addition of any one of the three substrates, cis-aconitate is the predominant species bound to Fea of the cluster along with solvent HxO and that cis-aconitate is bound in the citrate mode (carboxyl at C-2). The results with the nitro analogue show that the enzyme can also bind a substrate-like ligand to the cluster in the alternative isocitrate mode (carboxyl at C-1), as is implicit in models proposed for the aconitase reaction.

Fluorocitrate Ototoxicity

Fluorocitrate, an inhibitor of the tricarboxylic acid cycle at the aconitase reaction, produces a time and dose related neural dystrophy in the guinea pig cochlea. There is direct inhibition of succinic dehydrogenase activity but not nicotinamide adenine dinucleotide dehydrogenase and cytochrome oxidase via cytochrome c activities. The dystrophic neural changes morphologically are similar to those noted in primary neural degeneration and neural presbycusis in man. Neural degeneration in aging appears to be the result of a dissociation of biochemical reactions preventing the proper utilization of organic fuel molecules for generation of energy and direct or indirect inhibition of respiration.

METABOLISM OF C14 AMINO ACIDS AND AMIDES IN DETACHED LEAVES

Detached broad bean leaves were placed with their petioles in 0.01 M ammonium nitrate and allowed to carry on photosynthesis in C14O2 for various periods from 12 to 125 min. The radioactivities of the various amino acids formed from C14O2 were determined. In addition, these amino acids were degraded by decarboxylation with ninhydrin. From the specific activity data it was concluded that the amino acid closest to the site of carbon dioxide fixation in photosynthesis was alanine, followed by aspartic and glutamic acids, with the amides farthest removed. From the intramolecular distribution of label it was concluded that asparagine and glutamine were formed from their corresponding amino acids. The labelling in aspartic and glutamic acids was not consistent with the view that these two amino acids are formed from their corresponding α-keto acids produced by operation of the conventional tricarboxylic acid cycle. A C2 plus C2 condensation is postulated for the formation of aspartic acid. A shift in the double bond in the aconitase reaction of the tricarboxylic acid cycle would account for the observed labelling in glutamic acid. When acetate-1-C14 was fed to detached broad bean leaves in the light or dark, the distribution of label in glutamic acid supported the suggestion that there is such a. shift in the double bond in the aconitase reaction. Sodium arsenite, infiltrated into tobacco leaves, inhibited the biosynthesis of asparagine but not that of glutamine.