Isolation and properties of watermelon isocitrate lyase

The glyoxylate cycle enzyme, isocitrate lyase (EC 4.1.3.1) was purified from cotyledons of Citrullus vulgaris (watermelon). The final preparation, which had been 97-fold purified with a specific activity of 16.1 units/mg protein in a yield of 36%, was homogeneous by gel- and immunoelectrophoretic criteria. The tetrameric enzyme had: a molecular weight of 277 000, a sedimentation coefficient of 12.4 s, and a K_m for D_s-isocitrate equal to 0.25 mM. Isocitrate lyase from this source is not a glycoprotein as shown by total carbohydrate content after precipitation by trichloroacetic acid of the purified enzyme. Reduction of the enzyme with thiols increased activity and maximal activity was obtained with at least 5 mM dithiothreitol. EDTA partially substituted for thiol in freshly isolated enzyme. Watermelon isocitrate lyase was also protected against thermal denaturation at 60° for at least 1 hr by 5 mM Mg²⁺ plus 5 mM oxalate. Oxalate was a competitive inhibitor with respect to isocitrate (K_i: 1.5 μM, pH 7.5, 30°).     

Purification and Characterization of Isocitrate Lyase from Ethanol-grown Euglena gracilis

Isocitrate lyase was purified to homogeneity from ethanol-grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS-polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The K_m values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis.     

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.     

Isocitrate lyase from Pseudomonas indigofera: pH dependence of catalysis and binding of substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomonas indigofera was dependent on two dissociable groups (pK_a's of 6.9 and 8.6). The pH dependence of the pK_i for succinate, a product of isocitrate cleavage, implied that a dissociable group (pK_a of 6.0) on the enzyme functions in binding succinate. The pK_i's for maleate and itaconate (succinate analogs) were similarly pH dependent. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the pK_i's of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the pK_m for isocitrate and the pK_i for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme (pK_a of 5.8). The pH dependence of the pK_i for homoisocitrate was similar. In addition the K_i for succinate and K_m for isocitrate were dependent upon Mg²⁺ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofera, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (pK_a of 5.8) and one on phosphoenolpyruvate (pK_a of 6.35), contributed to the pH dependence observed with phosphoenolpyruvate.     

Methylamine metabolism in a pseudomonas species

The mechanism by which a nonphotosynthetic bacterium Pseudomonas sp. (Shaw Strain MA) grows on the one-carbon source, methylamine, was investigated by comparing enzyme levels of cells grown on methylamine, to cells grown on acetate or succinate. Cells grown on methylamine have elevated levels of the enzymes serine hydroxymethyl transferase, serine dehydratase, malic enzyme, glycerate dehydrogenase and malate lyase (CoA acetylating ATP-cleaving). These enzymes, in conjunction with a constitutive glyoxylate transaminase, can account for the net conversion of two one-carbon units into acetyl CoA. Cells grown on acetate or methylamine, but not succinate, contain the enzyme isocitrate lyase; while cells grown on acetate or succinate, but not methylamine, contain significant levels of malate synthetase. These findings suggest that the acetyl CoA derived from one-carbon units in methylamine grown cells, condenses with oxalacetate to yield citrate and then isocitrate, followed by cleavage to succinate and glyoxylate. Thus, growth on methylamine is accomplished by the net synthesis of succinate from two molecules of methyamine and two molecules of CO₂.     

Inhibition of isocitrate lyase from Pseudomonas indigofera by itaconate

The effect of the inhibitor itaconate on the activity of purified isocitrate lyase from Pseudomonas indigofera was examined for the reaction in both directions. Itaconate was found to equilibrate very slowly with its enzyme-bound form, so that a rapid change in itaconate concentration produced a gradual change in reaction velocity which eventually reached a new steady state. Kinetic studies of this relaxation phenomenon indicated that itaconate inhibited by binding the enzyme only after prior binding of glyoxylate, thus mimicking the kinetic behavior of succinate. On the basis of these studies, the dissociation constants for itaconate and glyoxylate from their respective enzyme-bound forms were calculated. More than half of the isocitrate lyase was complexed by glyoxylate during cleavage of saturating isocitrate. The rate constant for release of itaconate from the enzyme was calculated to be about 0.2 min^?1. Direct binding of [¹⁴C]itaconate and [¹⁴C]succinate to isocitrate lyase at pH 6.8 was measured. Some binding of both ligands was found in the absence of glyoxylate, which was stimulated by the presence of 1 mm glyoxylate. These results suggest that there are up to three or more binding sites per active subunit, but that only one of these is catalytic.     

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.     

Purification and properties of isocitrate lyase from flax seedlings.

Isocitrate lyase has been purified from flax (Linum usitatissimum) seedlings. The final preparation was homogeneous by the criteria of polyacrylamide disc gel electrophoresis, immunodiffusion, and immunoelectrophoresis. From exclusion chromatography on Sephadex G-200, the molecular weight and Stoke's radius of the enzyme were 264,000 and 5.28 × 10^?7 cm, respectively. The subunit molecular weight was 67,000. Thus, the enzyme appears to be tetrameric. The enzyme required Mg²⁺ and cysteine for activity. The optimal pH of the enzyme was 7.5 both in Tris and in phosphate buffers. There are three disulfide bridges and two of eight cysteine residues are buried. Inactivation of isocitrate lyase resulted from short-term modification of enzymatic thiols but this could be reversed by added thiols. The enzyme was competitively inhibited by glyoxylate, l-tartrate, and malonate in catalysis of isocitrate cleavage.     

Subcellular localization and properties of glyoxylate cycle enzymes in the liver of rats with alloxan diabetes.

Key enzymes of the glyoxylate cycle (isocitrate lyase and malate synthetase) were found in the liver and kidney of rats suffering from alloxan diabetes. The activities of these enzymes in the liver were 0.080 and 0.0430 U/mg protein, respectively. Isocitrate lyase activity in the kidney was 0.030 U/mg protein, and that of the malate synthetase was 0.018 U/mg protein. Peroxisomal localization of the enzymes was shown. A novel malate dehydrogenase isoform was found in a liver of rats suffering from the alloxan diabetes. The isocitrate lyase was isolated by selective (NH4)2SO4 precipitation and DEAE-Toyopearl chromatography. The resulting enzyme preparation had specific activity 6.1 U/mg protein, corresponding to 76.25-fold purification with 32.6% yield. The isocitrate lyase was found to follow the Michaelis--Menten kinetic scheme (Km for isocitrate, 0.08 mM) and to be competitively inhibited by glucose 1-phosphate (Ki = 1. 25 mM), succinate (Ki = 1.75 mM), and citrate (Ki = 1.0 mM); the pH optimum of the enzyme was 7.5 in Tris-HCl buffer.     

Modification of the active site of isocitrate lyase from Pseudomonas indigofera

Kinetic analysis of inactivation of isocitrate lyase from Pseudomonas indigofera by 3-bromopyruvate established that enzyme binds this compound prior to alkylation and that substrate, D_s-isocitrate, competes for the same site on the enzyme. The rate of inactivation was increased by EDTA which is a promoter of catalysis in the presence of activated (reduced) enzyme and substrate. The combination of products, glyoxylate plus succinate, also protected against inactivation. Glyoxylate plus itaconate, phosphoenolpyruvate, or maleate also protected. However, each of the latter three compounds or glyoxylate or succinate alone provided little or no protection. Pyruvate, a competitive inhibitor with respect to glyoxylate in the condensation reaction, also failed to protect. However, two dicarboxylates, meso-tartrate and oxalate, that are also competitive inhibitors with respect to glyoxylate provide some protection against inactivation by BrP perhaps by bridging across cationic sites that facilitate glyoxylate and succinate binding. These and other results imply that alkylation by 3-bromopyruvate occurs at the succinate part of the active site. A mechanism which includes a catalytic role for the cysteine residue at the active site is presented and discussed.     

Purification and Properties of Isocitrate Lyase from Pupas of the Butterfly Papilio machaon L.

Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (Ki = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 +/- 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer).     

Repression of oxalic acid-mediated mineral phosphate solubilization in rhizospheric isolates of Klebsiella pneumoniae by succinate

Two strains of Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate by secretion of oxalic acid from glucose. Activities of enzymes for periplasmic glucose oxidation (glucose dehydrogenase) and glyoxylate shunt (isocitrate lyase and glyoxylate oxidase) responsible for oxalic acid production were estimated. In presence of succinate, phosphate solubilization was completely inhibited, and the enzymes glucose dehydrogenase and glyoxylate oxidase were repressed. Significant activity of isocitrate lyase, the key enzyme for carbon flux through glyoxylate shunt and oxalic acid production during growth on glucose suggested that it could be inducible in nature, and its inhibition by succinate appeared to be similar to catabolite repression.     

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.     

GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE CYANOBACTERIUM ANACYSTIS NIDULANS1

The enzymes of the glyoxylate cycle, isocitrate lyase (EC.4.1.3.1) and malate synthase (EC.4.1.3.2), were measured in cell-free extracts from the cyanobacterium Anacystis nidulans Drouet during photoautotrophic growth in medium aerated with ordinary air (0.03% CO₂). Isocitrate lyase had an average specific activity of 112 nmoles·min^?1·mg protein^?1 whereas malate synthase had an average specific activity of 12.5 nmoles·min^?1·mg protein^?1. Unpurified isocitrate lyase showed classical Michaelis kinetics with a K_m of 8 mM. Isocitrate lyase activity was strongly inhibited by numerous cellular metabolites at 10 mM concentration. The previously reported low specific activity for isocitrate lyase may be due to metabolite inhibition caused by growth in high CO₂ concentrations. The activities reported for isocitrate lyase and malate synthase suggest the operation of the glyoxylate cycle in Anacystis nidulans under CO₂-limiting growth conditions.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

Properties of a Mg2+ independent isocitrate lyase from gamma irradiated preclimacteric banana (Musa cavendishii)

Isocitrate lyase (Ee 4·1,3·1) was purified seventy fold from gamma irradiated banana pulp tissue acetone powder. It showed an optimum pH of 6·0, and the Km value for DL-isocitrate was 0·8 mM. Among the various metabolic inhibitors, oxaloacetate was found to be the most potent and its inhibition was competitive. The enzyme activity was not dependent on externally added Mg²⁺. The Mg²⁺ content of the purified enzyme was 10–12 ng/rng protein. A method for the detection of the two multiple forms of isocitrate lyase present in this preparation was developed using 2,4-dinitrophenylhydrazine as detecting agent for glyoxylate formed during the isocitrate lyase reaction.     

Phosphorylation of Acinetobacter isocitrate lyase.

During growth on succinate, Acinetobacter calcoaceticus contains two forms of the enzyme isocitrate dehydrogenase. Addition of acetate to a lag-phase culture grown on succinate causes a dramatic increase in activity of form II of isocitrate dehydrogenase and in isocitrate lyase. Form II of isocitrate dehydrogenase may be responsible for the partition of isocitrate between the TCA cycle and the glyoxylate by-pass. This report describes the phosphorylation of the enzyme isocitrate lyase from A. calcoaceticus. This phosphorylation may be a regulatory mechanism for the glyoxylate by-pass.     

Isocitrate lyase in germinating spores of the fern Anemia phyllitidis

Isocitrate lyase was partially purified from germinating spores of the fern Anemia phyllitidis. The enzyme requires Mg²⁺ and thiol compounds for maximal activity and has a pH optimum between 6.5 and 7.5. The K_m of the enzyme for threo-Δ_s-isocitrate is 0.5 mM. Succinate inhibits the enzyme non-competitively (K_i. 1.8 mM). The increase of isocitrate lyase activity is closely correlated with the induction of the germination process. The fall of enzyme activity during germination is associated with the decline in triglyceride reserves.     

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.     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon