Itaconic acid: An inhibitor of isocitrate lyase in Tetrahymena pyriformis in vitro and in vivo

The succinate analog itaconic acid was observed to be a competitive inhibitor of the glyoxylate cycle specific enzyme isocitrate lyase (EC 4.1.3.1) in cell-free extracts of Tetrahymena pyriformis. Itaconic acid also inhibited net in vivo glycogen synthesis from glyoxylate cycle-dependent precursors such as acetate but not from glyoxylate cycle-independent precursors such as fructose. The effect of itaconic acid on the incorporation of ¹⁴C into glycogen from various ¹⁴C-labeled precursors was also consistent with inhibition of isocitrate lyase by this compound. Another analog of succinate which shares a common metabolic fate with itaconic acid, mesaconic acid, had no effect on isocitrate lyase activity in vitro or on ¹⁴C-labeled precursor incorporation into glycogen in vivo. In addition, itaconic acid did not affect gluconeogenesis from lactate in isolated perfused rat livers, a system lacking the enzyme isocitrate lyase. These results are taken as evidence that itaconic acid is an inhibitor of glyoxylate cycle-dependent glyconeogenesis Tetrahymena pyriformis via specific competitive inhibition of isocitrate lyase activity.     

2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes.

In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources ( approximately 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mM pyruvate, 150 mM succinate and 10 microM PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.     

On the mechanism of action of isocitrate lyase.

1. The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively. 2. Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein-containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl-S-enzyme but that of isocitrate lyase is not a corresponding succinyl-S-enzyme. 3. In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate. 4. Isotopic exchange experiments performed in H18-2O with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass-spectrometric analysis. 5. The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently-linked succinyl-enzyme, derived from this anhydride.     

Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli.

Vanadate was used as a substrate analogue to modify and subsequently localize active site serine residues of isocitrate lyase from Escherichia coli. Irradiation of the enzyme on ice with UV light in the presence of vanadate resulted in inactivation. Inactivation was prevented by the substrates glyoxylate or Ds-isocitrate and to a much lesser extent by succinate. Reduction of photoinactivated isocitrate lyase by NaBH4 partially restored enzyme activity. The photomodified enzyme was labeled by reduction with NaB[3H]4 in the presence and absence of the substrates succinate plus glyoxylate. Highly differential labeling of serine residues 319 and 321 in the absence of substrates suggests their importance in the action of isocitrate lyase. These residues are highly conserved in all five known sequences of this enzyme.     

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.     

Formation of enzyme-bound carbanion intermediate in the isocitrate lyase-catalyzed reaction: enzymatic reaction of tetranitromethane with substrates and its dependence on effector, pH, and metal ions

Isocitrate lyase of germinating castor seed endosperm catalyzes the reactions of succinate and of isocitrate (but not of glyoxylate) with tetranitromethane (TNM), giving rise to the nitroform anion (C-(NO2)3), analogous to the reaction of TNM with carbanions (O.P. Malhotra and U.N. Dwivedi, 1984, Ind. J. Biochem. Biophys. 21, 65-67). The kinetics of this reaction have been investigated under a variety of conditions. At a fixed TNM concentration, the initial rate of reaction exhibits a hyperbolic saturation of the enzyme with isocitrate. The reaction with succinate, however, shows "negative cooperativity" in succinate saturation and the data are consistent with the existence of two sets of succinate binding sites of unequal affinity ("tight" and "loose" sites). Equal reaction rates are observed at enzyme-saturating concentrations of succinate and isocitrate. In every case, the rate of reaction is proportional to the TNM concentration. In the presence of alpha-ketoglutarate, hyperbolic saturation curves are obtained for all the substrates (TNM and succinate or TNM and isocitrate). In the presence of this effector the Km of succinate and TNM are independent of the concentration of the second substrate. On the other hand, sets of parallel straight lines are obtained in the double-reciprocal plots for the enzymatic reaction of TNM with isocitrate in the presence of alpha-ketoglutarate. Studies on the effect of pH on the isocitrate lyase-catalyzed reactions of TNM with succinate, TNM with isocitrate, and succinate with glyoxylate in the absence as well as in the presence of alpha-ketoglutarate show that the proton behaves as an uncompetitive inhibitor in all these reactions, suggesting the presence of a "masked" basic group at the enzyme site, which is protonated in the presence of substrate only. The pKa value of this group lies in the range 6.7-6.9. The enzymatic reactions of TNM with succinate and isocitrate exhibit identical Mg2+ ion dependence. From a comparison of the data on the enzymatic reactions of TNM with the corresponding results on the physiological reaction catalyzed by this enzyme, it has been suggested that an ion pair intermediate (E+ X S-, in which E, S, and S- stand for enzyme, succinate, and succinate carbanion, respectively) lies on the pathway of catalysis by isocitrate lyase.     

Crystal structure of 2-methylisocitrate lyase (PrpB) from Escherichia coli and modelling of its ligand bound active centre

Following acetate, propionate is the second most abundant low molecular mass carbon compound found in soil. Many microorganisms, including most, if not all fungi, as well as several aerobic bacteria, such as Escherichia coli and Salmonella enterica oxidize propionate via the methylcitrate cycle. The enzyme 2-methylisocitrate lyase (PrpB) from Escherichia coli catalysing the last step of this cycle, the cleavage of 2-methylisocitrate to pyruvate and succinate, was crystallised and its structure determined to a resolution of 1.9A. The enzyme, which strictly depends on Mg(2+) for catalysis, belongs to the isocitrate lyase protein family. A common feature of members of this enzyme family is the movement of a so-called "active site loop" from an open into a closed conformation upon substrate binding thus shielding the reactants from the surrounding solvent. Since in the presented structure, PrpB contains, apart from a Mg(2+), no ligand, the active site loop is found in an open conformation. This conformation, however, differs significantly from the open conformation present in the so far known structures of ligand-free isocitrate lyases. A possible impact of this observation with respect to the different responses of isocitrate lyases and PrpB upon treatment with the common inhibitor 3-bromopyruvate is discussed. Based on the structure of ligand-bound isocitrate lyase from Mycobacterium tuberculosis a model of the substrate-bound PrpB enzyme in its closed conformation was created which provides hints towards the substrate specificity of this enzyme.     

Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308.

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.     

Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination.

By a newly developed double-stranded mutagenesis technique, histidine (H), glutamate (E), arginine (R) and leucine (L) have been substituted for the lysyl 193 residue (K-193) in isocitrate lyase from Escherichia coli. The substitutions for this residue, which is present in a highly conserved, cationic region, significantly affect both the Km for Ds-isocitrate and the apparent kcat of isocitrate lyase. Specifically, the conservative substitutions, K-193-->H (K193H) and K193R, reduce catalytic activity by ca. 50- and 14-fold, respectively, and the nonconservative changes, K193E and K193L, result in assembled tetrameric protein that is completely inactive. The K193H and K193R mutations also increase the Km of the enzyme by five- and twofold, respectively. These results indicate that the cationic and/or acid-base character of K193 is essential for isocitrate lyase activity. In addition to the noted effects on enzyme activity, the effects of the mutations on growth of JE10, an E. coli strain which does not express isocitrate lyase, were observed. Active isocitrate lyase is necessary for E. coli to grow on acetate as the sole carbon source. It was found that a mutation affecting the activity of isocitrate lyase similarly affects the growth of E. coli JE10 on acetate when the mutated plasmid is expressed in this organism. Specifically, the lag time before growth increases over sevenfold and almost twofold for E. coli JE10 expressing the K193H and K193R isocitrate lyase variants, respectively. In addition, the rate of growth decreases by almost 40-fold for E. coli JE10 cells expressing form K193H and ca. 2-fold for those expressing the K193R variants. Thus, the onset and rate of E. coli growth on acetate appears to depend on isocitrate lyase activity.     

Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli

The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306. Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not. Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase. The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.     

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.     

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.     

Control of isocitrate lyase in Nocardia salmonicolor (NCIB9701).

Nocardia salmonicolor, grown on acetate, commercial D,L-lactate or hydrocarbon substrates, has high isocitrate lyase activities compared with those resulting from growth on other carbon sources. This presumably reflects the anaplerotic role of the glyoxylate cycle during growth on the former substrates. Amongst a variety of compounds tested, including glucose, pyruvate and tricarboxylic acid cycle intermediates, only succinate and fumarate prevented an increase in enzyme activity in the presence of acetate. When acetate (equimolar to the initial sugar concentration) was added to cultures growing on glucose, there followed de novo synthesis of isocitrated lyase and isocitrate dehydrogenase, with increases in growth rate and glucose utilization, and both acetate and glucose were metabolized simultaneously. A minute amount of acetate (40 muM) caused isocitrate lyase synthesis (a three-fold increase in activity within 3 min of addition) when added to glucose-limited continuous cultures, but even large amounts added to nitrogen-limited batch cultures were ineffective. Malonate, at a concentration that was not totally growth-inhibitory (1mM) prevented the inhibition of acetate-stimulated isocitrate lyase synthesis by succinate, but fumarate still inhibited in the presence of malonate. Phosphoenolpyruvate is a non-competitive inhibitor of the enzyme (apparent Ki 1-7 mM). The results are consistent with the induction of isocitrate or a closely related metabolite, and catabolite repression by a C-4 acid of the tricarboxylic acid cycle, possibly fumarate.     

Effect of glucose and cyclic adenosine 3',5'-monophosphate on the synthesis of succinate dehydrogenase and isocitrate lyase in Escherichia coli.

Repressed respiration of Escherichia coli cells grown in the presence of 2% glucose was derepressed when the cells were incubated in a buffer containing casamino acids. The glucose-repressed cells were deficient in succinate dehydrogenase [EC 1.3.99.1] and isocitrate lyase [EC 4.1.3.1] activities, which increased during the incubation. The increases in respiratory activity and enzyme activity on incubation were repressed by glucose, but except for isocitrate lyase these repressions could be restored by the addition of cyclic adenosine 3',5'-monophosphate. Inhibitors of protein synthesis blocked the increase of enzyme activity on incubation without glucose, or with glucose and the cyclic nucleotide.     

Phosphorylation of isocitrate lyase in Escherichia coli

Isocitrate lyase from Escherichia coli becomes phosphorylated in vitro by an endogenous kinase when partially purified extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with histidine modifying reagents, and alkaline hydrolysis of in vitro phosphorylated enzyme indicated the presence of a phosphohistidine residue. Phosphorylation of isocitrate lyase can also occur in vivo, which indicates a possible regulatory significance of this modification. In addition to phosphorylation, isocitrate lyase is capable of incorporating label from both [alpha-32P]ATP and [14C]ATP suggesting that more than one type of covalent modification occurs on this enzyme. This report reviews the studies which have demonstrated the phosphorylation and modification of isocitrate lyase from Escherichia coli.     

Isocitrate lyase from flax : terminal residues, composition, active site, and catalysis

The cleavage of D_s-isocitrate catalyzed by isocitrate lyase from Linum usitatissimum results in the ordered release of succinate and glyoxylate. The glyoxylate analog 3-bromopyruvate irreversibly inactivates the flax enzyme in a process exhibiting saturation kinetics and protection by glyoxylate or isocitrate or the competitive inhibitor l-tartrate. Succinate provides considerably less protection. Results with 3-bromopyruvate suggest that this reagent modifies plant and prokaryotic isocitrate lyases differently. Treatment of the tetrameric 264,000-dalton flax enzyme with carboxypeptidase A results in a release of one histidine/subunit which is concordant with loss of activity. The only N-terminal residue is methionine. Treatment of flax enzyme with diethylpyrocarbonate at pH 6.5 selectively modifies two histidines per 67,000-dalton subunit. The reaction of one histidine residue is abolished by the binding of l-tartrate and the modification of one is coincident with inactivation. The carboxy-terminal and active-site modifications establish that one histidine residue/monomer is essential in the flax enzyme and considerably extend information heretofore available only for fungal and bacterial isocitrate lyase.     

Purification of isocitrate lyase from Escherichia coli and watermelon using fast protein liquid chromatography

The enzyme isocitrate lyase has been purified to gel electrophoretic homogeneity from Escherichia coli and watermelon. From cotyledons of the latter source, the enzyme is obtained in less than 8 hours after precipitation with (NH4)2 SO4 followed by fractionation on cationic Mono S microbeads and anionic Mono Q microbeads using Fast Protein Liquid Chromatography (FPLC). From a genetically engineered E. coli strain, in which high-level expression of isocitrate lyase occurs, the enzyme has been purified in one step from the high-speed supernatant using a Mono Q column with FPLC. These purifications, both of which give satisfactory yields, potentiate rapid access to isocitrate lyase from both prokaryotic and eukaryotic sources.     

Crystal structure of Salmonella typhimurium 2-methylisocitrate lyase (PrpB) and its complex with pyruvate and Mg(2+)

Propionate metabolism in Salmonella typhimurium occurs via 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (PrpB). Here we report the X-ray crystal structure of the native and the pyruvate/Mg(2+) bound PrpB from S. typhimurium, determined at 2.1 and 2.3A, respectively. The structure closely resembles that of the Escherichia coli enzyme. Unlike the E. coli PrpB, Mg(2+) could not be located in the native Salmonella PrpB. Only in pyruvate bound PrpB structure, Mg(2+) was found coordinated with pyruvate. Binding of pyruvate to PrpB seems to induce movement of the Mg(2+) by 2.5A from its position found in E. coli native PrpB. In both the native enzyme and pyruvate/Mg(2+) bound forms, the active site loop is completely disordered. Examination of the pocket in which pyruvate and glyoxalate bind to 2-methylisocitrate lyase and isocitrate lyase, respectively, reveals plausible rationale for different substrate specificities of these two enzymes. Structural similarities in substrate and metal atom binding site as well as presence of similar residues in the active site suggest possible similarities in the reaction mechanism.     

The inhibition of isocitrate lyase fromEscherichia coli by glyoxylate

Inhibition patterns have been studied to shed light on the current controversy involving the kinetic mechanism for isocitrate lyase fromEscherichia coli. A new coupled enzymatic assay for the product succinate has been developed, enabling the determination that glyoxylate, the other product, is a linear competitive inhibitor of isocitrate cleavage. This and other evidence suggest that the kinetic mechanism is steady-state, ordered uni-bi, and that succinate and glyoxylate are sequentially released from the enzyme after cleavage of isocitrate.     

Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli.

A structural gene for isocitrate lyase was isolated from a cosmid containing an ace locus of the Escherichia coli chromosome. Cloning and expression under control of the tac promoter in a multicopy plasmid showed that a 1.7-kilobase-pair DNA segment was sufficient for complementation of an aceA deletion mutation and overproduction of isocitrate lyase. DNA sequence analysis of the cloned gene and N-terminal protein sequencing of the cloned and wild-type enzymes revealed an entire aceA gene which encodes a 429-amino-acid residue polypeptide whose C-terminus is histidine. The deduced amino acid sequence for the 47.2-kilodalton subunit of E. coli isocitrate lyase could be aligned with that for the 64.8-kilodalton subunit of the castor bean enzyme with 39% identity except for limited N- and C-terminal regions and a 103-residue stretch that was unique for the plant enzyme and started approximately in the middle of that peptide.