RESUMEN
A four-stage purification procedure including ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose has been elaborated for isolation of isocitrate lyase (EC 4.1.3.1) isoforms from the cotyledons of soybean Glycine max L. Electrophoretically homogeneous preparations of two forms of the enzyme with specific activity of 5.28 and 5.81 U/mg protein have been obtained. Comparison of physicochemical, kinetic, and regulation characteristics of the isoforms obtained revealed fundamental differences between them. Thus, the isoform that migrated quickly in PAAG had a much lower affinity to isocitrate (K(M) - 50 microM) than the slowly migrating form (K(M) - 16 microM). It has been shown that the conservation of activity of the isoforms obtained depends on the presence of divalent cations (Mn2+ and Mg2+) in the medium. It is suggested to use the isoforms of isocitrate lyase isolated from soybeans for the development of biosensors for biochemical and kinetic assays.
Asunto(s)
Cotiledón/enzimología , Glycine max/enzimología , Isocitratoliasa/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Sulfato de Amonio/química , Técnicas Biosensibles , Cromatografía DEAE-Celulosa , Cromatografía por Intercambio Iónico , Isocitratoliasa/química , Isocitratos/química , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Proteínas de Plantas/química , Especificidad por SustratoRESUMEN
Tuberculosis (TB) is one of the leading causes of death worldwide. Long duration of TB therapy, results in the persistence and development of drug resistant strains of causative organism Mycobacterium tuberculosis (Mtb). Novel drug targets against persistent Mtb is an immediate need for overcoming this global menace. Isocitrate lyase (ICL), the first enzyme of glyoxylate pathway, is essential for persistent Mtb and absent in humans, hence a propitious target for drug development. Pathogenic Mtb H37Rv, have two types of ICLs - ICL1 encoded by icl (Rv0467) is well characterized and homologous to eubacterial enzyme whereas ICL2 encoded by aceA is more related to eukaryotic isocitrate lyase. To compound it, the aceA gene is split into two ORFs namely rv1915/aceAa and rv1916/aceAb. No translational product has been reported for the later and therefore, in vivo existence of Rv1916/ICL2b is debatable. This study reports recombinant production of Rv1916 in heterologous host E. coli BL21 (DE3) for structure function studies. The studies categorically demonstrate that akin to Mtb ICL1, recombinant Rv1916 also possess dual ICL and methylisocitrate lyase (MICL) activities in vitro. Based on in silico analysis, a putative function linked to secondary metabolite synthesis is assigned to unique mycobacterial domain IV.
Asunto(s)
Proteínas Bacterianas/química , Isocitratoliasa/química , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Conformación Proteica , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Isocitratoliasa/genética , Isocitratoliasa/aislamiento & purificación , Isocitratoliasa/metabolismo , Cinética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Filogenia , Relación Estructura-Actividad Cuantitativa , Proteínas RecombinantesRESUMEN
Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate flux and anaplerotic replenishment of pool of substrate required for biosynthetic process in Mycobacterium tuberculosis is a potential drug target for the antituberculosis drugs. Divalent cations induce differential effect of activation and inhibition of MtbIcl functional activity. The study for the first time demonstrates that interaction of cations with MtbIcl results in differential modulation of the enzyme structure which is probably the underlying mechanism for differential modulation of functional activity of enzyme by divalent cations. The Mg(2+) and Mn(2+) ions act as activators of the enzyme and in their absence no enzymatic activity was observed. These cations do not induce any significant structural alteration in the enzyme as observed by far-UV CD and solvent denaturation studies using chaotropic salts. However, the thermal denaturation studies demonstrate that they do interact with the noncatalytic alpha/beta barrel core domain of the enzyme and destabilize it. The inhibitors Zn(2+) and Cd(2+) interact directly with the catalytic domain of the enzyme and unfold it as a result of which complete loss of the enzymatic activity is observed in their presence. The results obtained from the studies provide intriguing insight into the possible mechanism of divalent cation-induced changes in structure, function, and stability of MtbIcl.
Asunto(s)
Cationes Bivalentes/farmacología , Isocitratoliasa/química , Isocitratoliasa/metabolismo , Mycobacterium tuberculosis/enzimología , Dominio Catalítico , Dicroismo Circular , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Isocitratoliasa/aislamiento & purificación , Modelos Moleculares , Pliegue de Proteína , Sales (Química)/farmacología , Electricidad Estática , TemperaturaRESUMEN
Isocitrate lyase (ICL, EC 4.1.3.1) is commonly present in oil-rich seeds in catalyzing the cleavage of isocitrate to glyoxylate and succinate and plays an essential role in lipid metabolism and gluconeogenesis. When peanut kernels (Tainan 14) were germinated at 30 degrees C, the cotyledon ICL activities increased substantially in the initial 4 days, and the 4-day-germinated cotyledons were subjected to ICL purification by Tris-HCl buffer extraction, heat treatment at 55 degrees C for 1 h, (NH4)2SO4 fractionation at 25-35% saturation, DEAE-cellulose chromatography, and Sephacryl S-300 gel filtration. A single 64 kDa SDS-PAGE protein band was obtained with 7.7% recovery and 37.5-fold purity. It was identified as ICL by LC-MS/MS analyses and Mascot Search with 494 as the highest Probability Based Mowse Score (PBMS). On the basis of the sequence of the homologous ICL of Glycine max, 26% of the peptide sequences of the peanut ICL were identified. During gel filtration, separation of peanut catalase (identified by LC-MS/MS and Mascot Search with 405 as the highest PBMS) from peanut ICL was achieved. The highest measured peanut ICL enzymatic activities were obtained at 45 degrees C and pH 7.0-7.8, respectively. The enzyme activities were stable (>80%) as stored for 8 h at 30 degrees C, 15 days at 4 degrees C, or 60 days at -25 degrees C. As affected by the supplements in the reactants for activity determinations, ICL activity was not affected by glucose up to 4%, sucrose up to 5%, or ethanol up to 8.33%.
Asunto(s)
Arachis/enzimología , Isocitratoliasa/aislamiento & purificación , Isocitratoliasa/metabolismo , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Semillas/enzimologíaRESUMEN
The glyoxylate cycle comprising isocitrate lyase (ICL) and malate synthase (MS) is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, which encodes malate synthase has been previously cloned from Streptomyces clavuligerus NRRL 3585 and characterized. In this study, the aceA gene, encoding ICL from S. clavuligerus NRRL 3585, was obtained via genome walking experiments and PCR. The fully sequenced open reading frame encodes 436 amino acids with a deduced M(r) of 47.5 kDa, consistent with the observed M(r) (49-67.5 kDa) of most ICL enzymes reported so far. The cloned aceA gene was expressed in Escherichia coli BL21(lambdaDE3) cells, from which ICL was purified as a His-tagged product and its functionality demonstrated. Furthermore, the relationship between the carbon sources, growth and ICL activity in S. clavuligerus were investigated. Rapid growth was observed when the cells were cultured on 0.5% (w/v) glycerol, while delayed growth was observed when cells were grown on 0.5% (w/v) acetate. However, in both cases, high levels of ICL activity coincided with a cessation of growth, suggesting a late physiological role played by ICL in the natural host, S. clavuligerus.
Asunto(s)
Isocitratoliasa/genética , Streptomyces/genética , Proteínas Bacterianas , Clonación Molecular , ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Isocitratoliasa/biosíntesis , Isocitratoliasa/aislamiento & purificación , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Operón , Filogenia , Streptomyces/enzimologíaRESUMEN
Two different crystal forms of isocitrate lyase (ICL) from Escherichia coli have been grown following the chemical modification of the enzyme by either 3-bromopyruvate or ethyl mercuri thiosalicylate (EMTS), contrasting strongly with difficulties in obtaining ordered crystals of the native enzyme. Both crystal forms are obtained using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. The crystals diffract well and X-ray photographs of the crystals have established that they are in space groups C222(1) and P3(1) (or its enantiomorph P3(2), respectively. Considerations of the values of Vm and measurements on the crystal density indicate that the asymmetric unit of both crystals contains four subunits.
Asunto(s)
Escherichia coli/enzimología , Isocitratoliasa/química , Piruvatos/farmacología , Timerosal/farmacología , Cristalización , Isocitratoliasa/aislamiento & purificación , Sustancias Macromoleculares , Conformación Proteica , Difracción de Rayos XRESUMEN
Euglena gracilis induced glyoxylate cycle enzymes when ethanol was fed as a sole carbon source. We purified, cloned and characterized a bifunctional glyoxylate cycle enzyme from E. gracilis (EgGCE). This enzyme consists of an N-terminal malate synthase (MS) domain fused to a C-terminal isocitrate lyase (ICL) domain in a single polypeptide chain. This domain order is inverted compared to the bifunctional glyoxylate cycle enzyme in Caenorhabditis elegans, an N-terminal ICL domain fused to a C-terminal MS domain. Purified EgGCE catalyzed the sequential ICL and MS reactions. ICL activity of purified EgGCE increased in the existence of acetyl-CoA at a concentration of micro-molar order. We discussed the physiological roles of the bifunctional glyoxylate cycle enzyme in these organisms as well as its molecular evolution.
Asunto(s)
Euglena gracilis/enzimología , Isocitratoliasa/genética , Isocitratoliasa/fisiología , Malato Sintasa/genética , Malato Sintasa/fisiología , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Catálisis , Clonación Molecular , ADN Complementario/genética , Etanol/administración & dosificación , Euglena gracilis/química , Euglena gracilis/metabolismo , Concentración de Iones de Hidrógeno , Isocitratoliasa/aislamiento & purificación , Cinética , Malato Sintasa/aislamiento & purificación , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Isocitrate lyase (EC 4.1.3.1) catalyzes the reversible conversion of d-isocitrate to succinate and glyoxylate. It is usually associated with the glyoxylate cycle in glyoxysomes, although the non-glyoxysomal form has been reported and its relation to interconversion of organic acids outside the glyoxylate cycle suggested. We investigated the expression of two isocitrate lyase genes and activities of the glyoxysomal (ICL1) and cytosolic (ICL2) forms of isocitrate lyase in amaranth (Amaranthus caudatus L.) seedlings. Both forms were separated and purified. The cytosolic form had a low optimum pH (6.5) and was activated by Mn(2+) ions, while Mg(2+) was ineffective, and had a lower affinity to d, l-isocitrate (Km 63 µM) as compared to the glyoxysomal form (optimum pH 7.5, K(m) 45 µM), which was activated by Mg(2+). The highest ICL1 activity was observed on the 3rd day of germination; then the activity and expression of the corresponding gene decreased, while the activity of ICL2 and gene expression increased to the 7th day of germination and then remained at the same level. It is concluded that the function of ICL1 is related to the glyoxylate cycle while ICL2 functions independently from the glyoxylate cycle and interconverts organic acids in the cytosol.
Asunto(s)
Amaranthus/enzimología , Amaranthus/genética , Citosol/enzimología , Regulación de la Expresión Génica de las Plantas , Glioxisomas/enzimología , Isocitratoliasa/genética , Secuencia de Bases , Centrifugación por Gradiente de Densidad , ADN Complementario/genética , Electroforesis en Gel de Agar , Regulación Enzimológica de la Expresión Génica , Genes de Plantas , Germinación , Concentración de Iones de Hidrógeno , Isocitratoliasa/aislamiento & purificación , Isocitratoliasa/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Factor 1 de Elongación Peptídica/metabolismo , Plantones/enzimología , Fracciones Subcelulares/enzimologíaRESUMEN
The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of food-starved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K(m)(isocitrate) was 68 microM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K(m)(acetyl-CoA) of 0.2 microM, a K(m)(glyoxylate) of 3 mM and a pH optimum of 7.6.
Asunto(s)
Glioxilatos/metabolismo , Isocitratoliasa/biosíntesis , Hígado/enzimología , Malato Sintasa/biosíntesis , Inanición/enzimología , Animales , Centrifugación por Gradiente de Densidad , Inducción Enzimática , Glioxilatos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Isocitratoliasa/aislamiento & purificación , Cinética , Malato Sintasa/aislamiento & purificación , Microcuerpos/enzimología , RatasRESUMEN
Key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), have been detected in the liver of alloxan-treated rats. The activity of ICL in rat liver was 0.040 micromol/min/mg protein and the activity of MS was 0.022 micromol/min/mg protein. These enzymes were associated with the peroxisomal fraction. The activities of citrate synthase, malate synthase and malate dehydrogenase detected in the peroxisomal fraction were also increased by alloxan treatment. Isocitrate lyase was partially purified and displayed catalytic and regulatory properties similar to those of the enzyme isolated from the liver of starved rats (Popov, V.N. et al. (1996) FEBS Lett. 391, 87-90).
Asunto(s)
Aloxano/farmacología , Glioxilatos/metabolismo , Isocitratoliasa/metabolismo , Hígado/enzimología , Malato Sintasa/metabolismo , Microcuerpos/enzimología , Aconitato Hidratasa/metabolismo , Animales , Fraccionamiento Celular , Citrato (si)-Sintasa/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Concentración de Iones de Hidrógeno , Isocitratoliasa/antagonistas & inhibidores , Isocitratoliasa/aislamiento & purificación , Riñón , Cinética , Hígado/efectos de los fármacos , Malato Deshidrogenasa/metabolismo , Microcuerpos/efectos de los fármacos , Microcuerpos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Especificidad de Órganos , RatasRESUMEN
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.
Asunto(s)
Isocitratoliasa/metabolismo , Oxo-Ácido-Liasas/metabolismo , Plantas/enzimología , Isocitratoliasa/antagonistas & inhibidores , Isocitratoliasa/aislamiento & purificación , Cinética , Peso Molecular , Conformación ProteicaRESUMEN
The induction of glyoxylate cycle enzyme activities was revealed in the liver and other organs of starving rats. A five day deprivation of food was followed by the appearance of isocitrate lyase (ICL) and malate synthase activities and the increase of malate dehydrogenase (MDH) and citrate synthase activities. The induction of MDH was associated with the appearance of its new isoform with Rf 0.52. ICL activity was revealed in the liver, blood, pancreas, kidney, lungs, heart, and skeletal muscles of starving rats, reaching a peak on day 5 of food deprivation. No significant changes of blood glucose level in starving rats were revealed until day 9. A homogeneous ICL preparation with a specific activity of 12.4 IU per mg protein was obtained as the results of five-stage purification procedure.
Asunto(s)
Glioxilatos/metabolismo , Isocitratoliasa/biosíntesis , Inanición/metabolismo , Aconitato Hidratasa/biosíntesis , Animales , Citrato (si)-Sintasa/biosíntesis , Metabolismo Energético , Inducción Enzimática , Privación de Alimentos , Isocitratoliasa/química , Isocitratoliasa/aislamiento & purificación , Hígado/química , Hígado/enzimología , Malato Deshidrogenasa/biosíntesis , Malato Sintasa/biosíntesis , Peso Molecular , Especificidad de Órganos , Ratas , Inanición/enzimologíaRESUMEN
The role of isocitrate lyase (ICL) in the glyoxylate cycle and its necessity for persistence and virulence of Mycobacterium tuberculosis has been well described. Recent reports have alluded to an additional role for this enzyme in M. tuberculosis metabolism, specifically for growth on propionate. A product of beta-oxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the by-products of its metabolism via the methylcitrate cycle is vital due to their potentially toxic effects. Although the genome of M. tuberculosis encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methylisocitrate lyase (MCL). Detailed structural analysis of the MCL from Escherichia coli suggested that the differences in substrate specificity between MCLs and ICLs could be attributed to three conserved amino acid substitutions in the active site, suggesting an MCL signature. However, here we provide enzymatic evidence that shows that despite the absence of the MCL signature, ICL1 from M. tuberculosis can clearly function as a MCL. Furthermore, the crystal structure of ICL1 with pyruvate and succinate bound demonstrates that the active site can accommodate the additional methyl group without significant changes to the structure.
Asunto(s)
Proteínas Bacterianas/química , Citratos/metabolismo , Glioxilatos/metabolismo , Isocitratoliasa/química , Mycobacterium tuberculosis/enzimología , Proteínas Bacterianas/aislamiento & purificación , Liasas de Carbono-Carbono/metabolismo , Citrato (si)-Sintasa/metabolismo , Cristalografía , Ácidos Grasos/metabolismo , Isocitratoliasa/aislamiento & purificación , Modelos Moleculares , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Conformación Proteica , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismoRESUMEN
Invasive aspergillosis, caused by Aspergillus fumigatus, is a severe systemic infection in immunocompromised patients. New drug targets are required, since therapeutic treatment often fails and is hampered by severe side effects of antifungals. Enzymes of the glyoxylate bypass are potential targets, since they are absent in humans, but required for growth of Aspergillus on C2-generating carbon sources. The key enzyme isocitrate lyase (ICL) can be inhibited by 3-nitropropionate, both as a purified enzyme and within intact cells, whereas the latter inhibition upregulates ICL promoter activity. ICL was found in distinct subcellular structures within growing hyphae, but only under conditions requiring ICL activity. In contrast, ICL was constitutively found in conidia, suggesting a specific role during germination. Lipids, as potential substrates, were detected in conidia and macrophages. Additionally, germinating conidia within macrophages contain ICL, suggesting that the glyoxylate shunt might be a relevant target for development of antifungals.
Asunto(s)
Aspergillus fumigatus/enzimología , Regulación Fúngica de la Expresión Génica , Isocitratoliasa/biosíntesis , Animales , Fusión Artificial Génica , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Secuencia de Bases , Clonación Molecular , ADN de Hongos/química , ADN de Hongos/genética , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Hifa/química , Isocitratoliasa/efectos de los fármacos , Isocitratoliasa/aislamiento & purificación , Lípidos/análisis , Macrófagos/microbiología , Ratones , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Nitrocompuestos/farmacología , Regiones Promotoras Genéticas , Propionatos/farmacología , Análisis de Secuencia de ADN , Coloración y Etiquetado , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.
Asunto(s)
Isocitratoliasa/aislamiento & purificación , Oxo-Ácido-Liasas/aislamiento & purificación , Cromatografía DEAE-Celulosa , Calor , Isocitratoliasa/clasificación , Peso Molecular , PlantasRESUMEN
Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) was isolated from cotyledons of Lupinus seedlings, purified 100-fold with respect to its initial specific activity and characterized (Km, pH optimum, Mg2+ requirement, sulfhydryl inhibitors, and synthase activity). The final purified preparation consisted of two homogeneous protein bands clearly separated by electrophoresis on polyacrylamide gel and chromatography on Sephadex G 200. Reducing agents are necessary for the maintenance of enzyme activity. The most effective reducing agent studied was 1,4-dithioerythreitol. The effect of several metabolites (oxalate, malonate, phosphoenolpyruvate, succinate, malate, tartrate, gluconate-6-phosphate, sorbose, sorbitol, and inositol) on the activity of purified preparations was tested. Oxalate proved to be the strongest inhibitor, seconded closely by phosphoenolpyruvate. The spectral characteristics of the purified enzyme are as follows: ultraviolet peak at 280 nm and fluorescence peak at 340 nm. The solid state infrared spectrum of the enzyme (lyophilized) showed that the enzyme was mostly in the alpha-helix conformation with very slight random orientation.
Asunto(s)
Isocitratoliasa/metabolismo , Oxo-Ácido-Liasas/metabolismo , Plantas/enzimología , Cloromercuribenzoatos/farmacología , Isocitratoliasa/aislamiento & purificación , CinéticaRESUMEN
A new purification procedure for isocitrate lyase from Pinus pinea is reported. The final preparation shows charge homogeneity and a purity degree higher than 95%. It is possible to remove catalase completely by exploiting the high hydrophobicity of isocitrate lyase. The enzyme has a Mr of 264,000 and is likely composed of four subunits, each with a Mr of 66,000. The binding of radioactively labeled oxalate revealed four catalytic sites per oligomer. These data suggest that isocitrate lyase subunits are similar, if not identical. The Michaelis constant for isocitrate is equal to 33 microM; molecular activity is about 2670 mol X min-1 X mol of enzyme-1. The amino acid composition of the enzyme was also determined. Isocitrate lyase appears resistant to proteolysis by carboxypeptidase A. Hydrazinolysis, Edman degradation, and dansyl chloride treatment indicate that both carboxy and amino terminals are probably inaccessible or blocked.
Asunto(s)
Isocitratoliasa/aislamiento & purificación , Oxo-Ácido-Liasas/aislamiento & purificación , Plantas/enzimología , Aminoácidos/análisis , Sitios de Unión , Catálisis , Cromatografía/métodos , Electroforesis en Gel de Poliacrilamida , Cinética , Peso MolecularRESUMEN
Isocitrate lyase was isolated in homogeneous state from a thermophilic Bacillus. The enzyme has a mol.wt. of 180000 and a pI of 4.5 and contains threonine as the N-terminal residue. It resembles in size the cognate enzyme from the mesophilic bacterium Pseudomonas indigofera, but is smaller than the enzyme from the eukaryotic fungus Neurospora crassa. All three lyases are tetramers and similar in amino acid composition, but the thermophile enzyme is distinctive from its mesophilic coutnerparts in possessing a lower catalytic-centre activity, greater resistance to chemical and thermal denaturation and fewer thiol groups and in being strongly activated by salts. Salt activation, by 0.4M-KCl, is about 3-fold at 30 degrees C and pH 6.8 and weakens progressively as the temperature or pH is raised. The activation is probably due to a change in the enzyme conformation caused by the electrolyte modifying the interaction between charged groups or between hydrophobic groups in protein. The possible significance of the salt activation, of the relative paucity of thiol groups and of the greater resistance to chemical denaturants is discussed. Besides its effect on the Vmax., KCl produces large increases in the magnitude of several kinetic parameters. A rise in reaction temperature from 30 to 55 degrees C produces a somewhat similar result. In view of these peculiar features, the patterns of inhibition of enzyme activity by compounds such as succinate and phosphoenolpyruvate were examined at 30 and 55 degrees C in the presence and absence of KCl.
Asunto(s)
Bacillus/enzimología , Isocitratoliasa/aislamiento & purificación , Oxo-Ácido-Liasas/aislamiento & purificación , Aminoácidos/análisis , Fenómenos Químicos , Química , Electroforesis en Gel de Poliacrilamida , Isocitratoliasa/antagonistas & inhibidores , Cinética , Peso Molecular , Cloruro de PotasioRESUMEN
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.