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.     

Inhibiton of isocitrate dehydrogenase and isocitrate lyase from Acinetobacter calcoaceticus by acids of the citrate and glyoxylate cycle]

Acinetobacter calcoaceticus contains two forms of NADP+-dependent isocitrate dehydrogenases differing, among others, by their molecular weights and regulatory properties. The regulation of the high-molecular form of isocitrate dehydrogenase and of isocitrate lyase by organic acids, either belonging or related to the citrate and glyoxalate cycle, is investigated. While alpha-ketoglutarate and oxalacetate competitively inhibit the isocitrate dehydrogenase against Ds-isocitrate, glyoxylate and pyruvate were found to increase Vmax and to lower the KM value for Ds-isocitrate and NADP+. Simultaneous addition of oxalacetate and glyoxylate (not, however, addition of the nonenzymatically formed condensation product of both compound) nullified the activation of isocitrate dehydrogenase by glyoxylate, and potentiates the inhibitory effect of oxalacetate. Alpha-ketoglutarate, succinate, and phosphoenolpyruvate inhibit the isocitrate lyase in a noncompetitive fashion against DS-isocitrate; L-malate, oxalacetate and glyoxylate inhibit competitively. The intermediates of the citrate and glyoxylate cycle afford additive inhibition of the isocitrate lyase. The importance of organic acids of the citrate and glyoxylate cycle and of phosphoenolpyruvate for the regulation of the citrate and glyoxylate cycle at the level of isocitrate dehydrogenase and isocitrate lyase is discussed.     

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.     

Mitochondrial isocitrate dehydrogenase and isocitrate oxidation of rat ventral prostate.

Mitochondrial preparations isolated from rat ventral prostate were capable of oxidizing isocitrate by way of NADP isocitrate dehydrogenase (NADP-IDH) and NAD-IDH. NAD-IDH activity required ADP for activation. The pH responses for NAD-IDH and NADP-IDH were quite different. The results indicated that two different enzymes were involved in the NAD- and NADP-IDH activities. Indirect evidence indicated that NADPH-NAD transhydrogenase activity might also be involved in the mitochondrial pathway for isocitrate oxidation. NADP-IDH activity was significantly greater than NAD-IDH activity. The oxidation of isocitrate through IDH activity was coupled to the cytochrome system by NADPH- and NADH-cytochrome c reductase activities. Citrate, via isocitrate, oxidation proceeded at a much slower rate suggesting that aconitase activity could be limiting in the oxidation of citrate. In comparison to other tissues, the prostate oxidative enzyme activities are considerably lower. The results suggest that the accumulation of high prostate citrate levels is not due to a limitation imposed by a lack of IDH activity in prostate mitochondria.     

Role and control of isocitrate lyase in Candida lipolytica.

Mutants of Candida lipolytica that were unable to grow on acetate but able to utilize succinate or glycerol as a sole carbon source were isolated. Amongst the mutants isolated, one strain (Icl-) was specifically deficient in isocitrate lyase activity, whereas another strain (Acos-) was deficient in acetyl coenzyme A synthetase activity. Since the Icl- mutant could not grow either on n-alkane or its derivatives, such as fatty acid and long-chain dicarboxylic acid, any anaplerotic route other than the glyoxylate pathway was inconceivable as far as growth on these carbon sources was concerned. Acetyl coenzyme A is most likely a metabolic inducer of isocitrate lyase and malate synthase, because the Acos- mutant was characterized by the least susceptibility to induction of these enzymes by acetate. The structural gene for isocitrate lyase was most probably impaired in the Icl- mutant, since revertants (Icl-) produced thermolabile isocitrate lyase. The production of isocitrate from n-alkane by the revertants was enhanced in comparison with the parental strain.     

Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

Glucose-stimulated phosphorylation of yeast isocitrate lyase in vivo

Incorporation of 32P into Saccharomyces cerevisiae isocitrate lyase was observed after addition of glucose to a culture incubated with [32P]orthophosphoric acid. A band of 32P-labelled protein was coincident with the enzyme band when immunoprecipitates were subjected to SDS-PAGE and autoradiography. No label was found in the band corresponding to the isocitrate lyase when immunoprecipitation was done with a control pre-immune serum or in the presence of excess pure unlabelled enzyme. The incorporation of phosphate was associated with a decrease in enzyme activity. Phosphorylated isocitrate lyase was not proteolytically degraded when cells were cultured in mineral medium. The loss of protein antigenicity only took place when the yeast was grown in a complex medium containing glucose.     

Stimulation of isocitrate lyase biosynthesis by hydroxylamine and hydrazine

Summary Recently it has been demonstrated that hydroxylamine is an activator of triglyceride catabolism. We have studied the effect of hydroxylamine on isocitrate lyase activity and lipid catabolism and have noted a stimulation of isocitrate lyase biosynthesis by 5mm hydroxylamine. The specificity of this effect was tested with a number of representative enzymes of other metabolic pathways.In an attempt to study the possible mechanism of action of hydroxylamine we have also tested the effects of two substances that are structural or functional analogues of hydroxylamine, namely, ethanolamine and hydrazine, both on the enzyme level in plant cultures and on the activity of enzyme preparations.From our data we may conclude that de novo biosynthesis of isocitrate lyase depends on the reaction of hydroxylamine or hydrazine with glyoxylate to give the corresponding oxime and hydrazone. The removal of glyoxylate from the biological equilibrium in this way could cause extra formation of isocitrate lyase.     

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.     

Caenorhabditis elegans and Ascaris suum: fragmentation of isocitrate lyase in crude extracts

It is well known that proteolysis often occurs after rupture of metazoan cells. Thus proteins isolated from extracts may not be representative of their native cellular counterparts. In the present research, extensive proteolysis was observed in crude extracts of the freeliving soil nematode Caenorhabditis elegans and the parasitic nematode Ascaris suum. Phenylmethylsulfonyl fluoride (PMSF) reduced the loss in activity of isocitrate lyase (EC 4.1.3.1), fumarase (EC 4.2.1.2), and citrate synthase (EC 4.1.3.7) in extracts of C. elegans but had little or no effect upon loss of malate synthase (EC 4.1.3.2). Catalase (EC 1.11.1.6) was stable. The loss of isocitrate lyase and citrate synthase was less pronounced in extracts of 22-day-old embryos of A. suum. Catalase decayed in these extracts. The addition of PMSF reduced the loss in all three of these activities. Fumarase was stable. The number of active fragments of isocitrate lyase recovered after filtration on Sephadex G-200 increased with the length of storage of crude extracts in the absence of PMSF at 4 C. Even in the presence of PMSF five activity peaks were observed after storage of extracts of C. elegans at 4 C for 72 hr. The molecular weights of active species ranged between 549,000 and 128,000 for isocitrate lyase in extracts of either C. elegans or A. suum. The 549,000- and 214,000-dalton species of isocitrate lyase from A. suum were much more labile at 50 C than the 543,000- and 195,000-dalton species from C. elegans.     

Steady-state kinetic analysis of isocitrate lyase from Lupinus seeds: considerations on a possible catalytic mechanism of isocitrate lyase from plants

Isocitrate lyase catalyzes the reversible cleavage of isocitrate into glyoxylate and succinate. The kinetic mechanism of bacterial isocitrate lyase has been reported to be ordered uni-bi. Moreover, it has been proposed that isocitrate lyase in higher plants may be switched on and off by a succinylation/desuccinylation mechanism. Similarly to bacterial citrate lyase, in which an acetylation/deacetylation mechanism is operative, succinylation might also play a role in the catalytic mechanism of plant isocitrate lyase. We have investigated the kinetic mechanism of isocitrate lyase from Lupinus seeds. The results reported in this paper show that the system follows a preferentially ordered uni-bi pathway in which the succinate is released first. On the basis of our results and some other recently reported data, we conclude that it is unlikely that bacterial and plant isocitrate lyases have different catalytic mechanisms.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Kinetic regulation of yeast NAD-specific isocitrate dehydrogenase by citrate

The present results suggest that the enzyme modifier citrate and the substrate isocitrate are bound at different sites on yeast NAD-specific isocitrate dehydrogenase and that citrate diminishes the binding of the positive effector 5'-AMP, thereby causing a decreased rate of enzyme catalysis. This interpretation differs from the earlier proposal that citrate can replace isocitrate at an activator site on the enzyme and can cause inhibition by binding at its catalytic site [Atkinson et al. (1965) J. Biol. Chem. 240, 2682]. The present proposal is supported by the following observations: At constant subsaturating levels of isocitrate, NAD+, and Mg2+ without AMP, up to 10 mM citrate was an activator and not an inhibitor. Citrate decreased velocity for AMP-activated enzyme; however, with increasing citrate the specific activity with AMP asymptotically approached but did not decrease below the level of the enzyme maximally activated by citrate in the absence of AMP. When added singly, AMP decreased S0.5 for isocitrate without changing the Hill number (n), whereas citrate lowered n without changing S0.5 for isocitrate. The difference in action of these modifiers indicated that they were bound at separate sites on the enzyme. The binding of citrate appeared to cause a conformational change in the protein that lowered the enzyme's affinity for AMP. This was consistent with the findings that citrate (or the citrate agonist fluorocitrate) (i) resulted in an increase in S0.5 for isocitrate with the AMP-activated enzyme and (ii) decreased binding of the positive effector analogue TNP-AMP as measured by fluorescence change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiology and metabolism of two alkane oxidizing and citric acid producing strains of Candida parapsilosis

Summary The activity of enzymes of the tricarboxylic acid (TAC) and glyoxylate (GC) cycles in Candida parapsilosis (wild type KSh 21 and mutant 337) were studied under different physiological and metabolic conditions. C. parapsilosis differed in most of its enzyme activities from other non-citric acid producing yeasts. Furthermore, pH-value, temperature and age of culture proved to act differently on both strains of the tested organism.The addition of trans-aconitate increased not only the growth but also the activities of citrate synthase and some other enzymes while that of aconitase decreased enormously.The high citrate synthase activity might be connected with the role of citrate in the transport of acetyl groups.Abbreviations CS citrate synthase - AC aconitase - ICDH isocitrate dehydrogenase - GDH glutamate dehydrogenase - Fum fumarase - MDH malate dehydrogenase - ICL isocitrate lyase - MS malate synthase     

Acetate-nonutilizing Mutants of Neurospora crassa II. Biochemical Deficiencies and the Roles of Certain Enzymes

The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing (acu) mutants of Neurospora crassa, both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed.     

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.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.