RESUMEN
Transcriptional factor p53 is a master regulator of energy metabolism. Energy metabolism strongly depends on thiamine (vitamin B1) and/or its natural derivatives. Thiamine diphosphate (ThDP), which is a major thiamine derivative, affects p53 binding to DNA. In order to elucidate the mechanism of regulation of thiamine-dependent metabolism by p53, we assessed putative p53-binding sites near transcription starting points in genes coding for transporters and enzymes, whose function is associated with thiamine and/or its derivatives. The predictions were validated by studying cell metabolic response to the p53 inducer cisplatin. Expression of p53 and its known target, p21, has been evaluated in cisplatin-treated and control human lung adenocarcinoma A549 cells that possess functional p53 pathway. We also investigated the activity of enzymes involved in the thiamine-dependent energy metabolism. Along with upregulating the expression of p53 and p21, cisplatin affected the activities of metabolic enzymes, whose genes were predicted as carrying the p53-binding sites. The activity of glutamate dehydrogenase GDH2 isoenzyme strongly decreased, while the activities of NADP+-dependent isocitrate dehydrogenase (IDH) and malic enzymes, as well as the activity of 2-oxoglutarate dehydrogenase complex at its endogenous ThDP level, were elevated. Simultaneously, the activities of NAD+-dependent IDH, mitochondrial aspartate aminotransferase, and two malate dehydrogenase isoenzymes, whose genes were not predicted to have the p53-binding sequences near the transcription starting points, were upregulated by cisplatin. The p53-dependent regulation of the assayed metabolic enzymes correlated with induction of p21 by p53 rather than induction of p53 itself.
Asunto(s)
Tiamina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/metabolismo , Cisplatino/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Metabolismo Energético , Glutamato Deshidrogenasa/metabolismo , Humanos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Oxidación-Reducción , Tiamina Pirofosfato/metabolismoRESUMEN
Unlike the OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH), which is an essential enzyme present in all animal tissues, expression of the DHTKD1-encoded isoenzyme, 2-oxoadipate dehydrogenase (OADH), depends on a number of factors, and mutant DHTKD1 phenotypes are rarely manifested. Physiological significance of OADH is also obscured by the fact that both isoenzymes transform 2-oxoglutarate and 2-oxoadipate. By analogy with other members of the 2-oxo acid dehydrogenases family, OADH is assumed to be a component of the multienzyme complex that catalyzes oxidative decarboxylation of 2-oxoadipate. This study aims at molecular characterization of OADH from animal tissues. Phylogenetic analysis of 2-oxo acid dehydrogenases reveals OADH only in animals and Dictyostelium discoideum slime mold, within a common branch with bacterial OGDH. Examination of partially purified animal OADH by immunoblotting and mass spectrometry identifies two OADH isoforms with molecular weights of about 130 and 70 kDa. These isoforms are not observed upon the expression of human DHTKD1 protein in either bacterial or yeast system, where the synthesized OADH is of expected molecular weight (about 100 kDa). Thus, the OADH isoforms present in animal tissues, may result from the animal-specific regulation of the DHTKD1 expression and/or posttranslational modifications of the encoded protein. Mapping of the peptides identified in the OADH preparations, onto the protein structure suggests that the 70-kDa isoform is truncated at the N-terminus, but retains the active site. Since the N-terminal domain of OGDH is required for the formation of the multienzyme complex, it is possible that the 70-kDa isoform catalyzes non-oxidative transformation of dicarboxylic 2-oxo acids that does not require the multienzyme structure. In this case, the ratio of the OADH isoforms in animal tissues may correspond to the ratio between the oxidative and non-oxidative decarboxylation of 2-oxoadipate.
Asunto(s)
Encéfalo/metabolismo , Escherichia coli/metabolismo , Complejo Cetoglutarato Deshidrogenasa/química , Hígado/metabolismo , Miocardio/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Dominio Catalítico , Dictyostelium/genética , Dictyostelium/metabolismo , Escherichia coli/genética , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Masculino , Oxidación-Reducción , Filogenia , Ratas , Ratas Wistar , Saccharomyces cerevisiae/genéticaRESUMEN
Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian enzymes is incomplete. In this work, the protein fraction of bovine brain synaptosomes displaying phosphatase activity toward thiamine derivatives was subjected to affinity chromatography on thiamine-Sepharose. Protein fractions eluted with thiamine (pH 7.4 or 5.6), NaCl, and urea were assayed for the phosphatase activity against thiamine monophosphate (ThMP), thiamine diphosphate (ThDP), and structurally similar purine nucleotides. Proteins in each fraction were identified by mass spectrometry using the SwissProt database for all organisms because of insufficient annotation of the bovine genome. Peptides of two annotated bacterial phosphatases, alkaline phosphatase L from the DING protein family and exopolyphosphatase, were identified in the acidic thiamine eluate. The abundance of peptides of alkaline phosphatase L and exopolyphosphatase in the eluted fractions correlated with ThMPase and ThDPase activities, respectively. The elution profiles of the ThMPase and ThDPase activities differed from the elution profiles of nucleotide phosphatases, thus indicating the specificity of these enzymes toward thiamine derivatives. The search for mammalian DING phosphatases in the eluates from thiamine-Sepharose revealed X-DING-CD4, mostly eluted by the acidic thiamine solution (pH 5.6). The identified exopolyphosphatase demonstrated structural similarity with apyrases possessing the ThDPase activity. The obtained results demonstrate that mammalian DING proteins and apyrases exhibit ThMPase and ThDPase activity, respectively.
Asunto(s)
Encéfalo/enzimología , Monoéster Fosfórico Hidrolasas/química , Sinaptosomas/enzimología , Tiamina/química , Animales , Dominio Catalítico , Bovinos , Cromatografía de Afinidad , Difosfatos/química , Genoma , Humanos , Concentración de Iones de Hidrógeno , Especificidad por Sustrato , Tiamina Monofosfato/química , Tiamina Pirofosfato/química , Urea/químicaRESUMEN
To study the mechanisms of the non-coenzyme action of thiamine and its diphosphate (ThDP) on brain proteins, proteins of acetone extract of bovine brain synaptosomes or the homogenate of rat brain cortex were subjected to affinity chromatography on thiamine-modified Sepharose. In the step-wise eluates by thiamine (at pH 7.4 or 5.6), NaCl, and urea, the occurrence of glutamate dehydrogenase (GDH) and isoenzymes of malate dehydrogenase (MDH) along with the influence of thiamine and/or ThDP on the enzymatic activities were characterized using mass spectrometry and kinetic experiments. Maximal activation of the malate dehydrogenase reaction by thiamine is observed after the protein elution with the acidic thiamine solution, which does not elute the MDH1 isoenzyme. Effects of exogenous thiamine or ThDP on the GDH activity may depend on endogenous enzyme regulators. For example, thiamine and/or ThDP activate the brain GDH in eluates from thiamine-Sepharose but inhibit the enzyme in the crude preparations applied to the sorbent. Inhibition of GDH by ThDP is observed using the ADP-activated enzyme. Compared to the affinity chromatography employing the elution by thiamine at pH 7.4, the procedure at pH 5.6 decreases the activation of GDH by thiamine (but not ThDP) in the eluates with NaCl and urea. Simultaneously, the MDH2 content and total GDH activity are higher after the affinity elution at pH 5.6 than at pH 7.4, suggesting the role of the known interaction of GDH with MDH2 in stabilizing the activity of GDH and in the regulation of GDH by thiamine. The biological potential of thiamine-dependent regulation of the brain GDH is confirmed in vivo by demonstration of changes in regulatory properties of GDH after administration of a high dose of thiamine to rats. Bioinformatics analysis of the thiamine-eluted brain proteins shows a specific enrichment of their annotation terms with "phosphoprotein", "acetylation", and "methylation". The relationship between thiamine and the posttranslational modifications in brain may contribute to the neuroprotective effects of high doses of thiamine, including the regulation of oxidation of the major excitatory neurotransmitter in brain - glutamate.
Asunto(s)
Encéfalo/enzimología , Glutamato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , Tiamina Pirofosfato/farmacología , Tiamina/farmacología , Animales , Bovinos , Activación Enzimática , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
Thiamine (vitamin B1) is a precursor of the well-known coenzyme of central metabolic pathways thiamine diphosphate (ThDP). Highly intense glucose oxidation in the brain requires ThDP-dependent enzymes, which determines the critical significance of thiamine for neuronal functions. However, thiamine can also act through the non-coenzyme mechanisms. The well-known facilitation of acetylcholinergic neurotransmission upon the thiamine and acetylcholine co-release into the synaptic cleft has been supported by the discovery of thiamine triphosphate (ThTP)-dependent phosphorylation of the acetylcholine receptor-associated protein rapsyn, and thiamine interaction with the TAS2R1 receptor, resulting in the activation of synaptic ion currents. The non-coenzyme regulatory binding of thiamine compounds has been demonstrated for the transcriptional regulator p53, poly(ADP-ribose) polymerase, prion protein PRNP, and a number of key metabolic enzymes that do not use ThDP as a coenzyme. The accumulated data indicate that the molecular mechanisms of the neurotropic action of thiamine are far broader than it has been originally believed, and closely linked to the metabolism of thiamine and its derivatives in animals. The significance of this topic has been illustrated by the recently established competition between thiamine and the antidiabetic drug metformin for common transporters, which can be the reason for the thiamine deficiency underlying metformin side effects. Here, we also discuss the medical implications of the research on thiamine, including the role of thiaminases in thiamine reutilization and biosynthesis of thiamine antagonists; molecular mechanisms of action of natural and synthetic thiamine antagonists, and biotransformation of pharmacological forms of thiamine. Given the wide medical application of thiamine and its synthetic forms, these aspects are of high importance for medicine and pharmacology, including the therapy of neurodegenerative diseases.
Asunto(s)
Hipoglucemiantes/metabolismo , Metformina/metabolismo , Tiamina/análogos & derivados , Tiamina/metabolismo , Complejo Vitamínico B/metabolismo , Animales , Encéfalo/metabolismo , Coenzimas , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/efectos adversos , Metformina/administración & dosificación , Metformina/efectos adversos , Ratones , Fosforilación , Transporte de Proteínas/fisiología , Ratas , Tiamina/efectos adversos , Tiamina/farmacología , Deficiencia de Tiamina/etiología , Deficiencia de Tiamina/prevención & control , Tiamina Pirofosfato/metabolismo , Complejo Vitamínico B/efectos adversos , Complejo Vitamínico B/farmacologíaRESUMEN
An optimized method for analysis of free amino acids using a modified lithium-citrate buffer system with a Hitachi L-8800 amino acid analyzer is described. It demonstrates clear advantages over the sodium-citrate buffer system commonly used for the analysis of protein hydrolysates. A sample pretreatment technique for amino acid analysis of brain extracts is also discussed. The focus has been placed on the possibility of quantitative determination of the reduced form of glutathione (GSH) with simultaneous analysis of all other amino acids in brain extracts. The method was validated and calibration coefficient (KGSH) was determined. Examples of chromatographic separation of free amino acids in extracts derived from different parts of the brain are presented.
Asunto(s)
Aminoácidos/análisis , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Aminoácidos/aislamiento & purificación , Animales , Cromatografía por Intercambio Iónico , Citratos/química , Glutatión/análisis , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Molecular mechanisms of long-term changes in brain metabolism after thiamine administration (single i.p. injection, 400 mg/kg) were investigated. Protocols for discrimination of the activities of the thiamine diphosphate (ThDP)-dependent 2-oxoglutarate and 2-oxoadipate dehydrogenases were developed to characterize specific regulation of the multienzyme complexes of the 2-oxoglutarate (OGDHC) and 2-oxoadipate (OADHC) dehydrogenases by thiamine. The thiamine-induced changes depended on the brain-region-specific expression of the ThDP-dependent dehydrogenases. In the cerebral cortex, the original levels of OGDHC and OADHC were relatively high and not increased by thiamine, whereas in the cerebellum thiamine upregulated the OGDHC and OADHC activities, whose original levels were relatively low. The effects of thiamine on each of the complexes were different and associated with metabolic rearrangements, which included (i) the brain-region-specific alterations of glutamine synthase and/or glutamate dehydrogenase and NADP+-dependent malic enzyme, (ii) the brain-region-specific changes of the amino acid profiles, and (iii) decreased levels of a number of amino acids in blood plasma. Along with the assays of enzymatic activities and average levels of amino acids in the blood and brain, the thiamine-induced metabolic rearrangements were assessed by analysis of correlations between the levels of amino acids. The set and parameters of the correlations were tissue-specific, and their responses to the thiamine treatment provided additional information on metabolic changes, compared to that gained from the average levels of amino acids. Taken together, the data suggest that thiamine decreases catabolism of amino acids by means of a complex and long-term regulation of metabolic flux through the tricarboxylic acid cycle, which includes coupled changes in activities of the ThDP-dependent dehydrogenases of 2-oxoglutarate and 2-oxoadipate and adjacent enzymes.
Asunto(s)
Aminoácidos/metabolismo , Corteza Cerebral/enzimología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Cetona Oxidorreductasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Tiamina/farmacología , Animales , Femenino , Ratas , Ratas Sprague-DawleyRESUMEN
Decreased thiamine and reduced activity of thiamine diphosphate (ThDP)-dependent 2-oxoglutarate dehydrogenase (OGDH) cause neurodegeneration. We hypothesized on concerted cell-specific regulation of the thiamine metabolism and ThDP-dependent reactions. We identified a smaller thiamine pool, a lower expression of the mitochondrial ThDP transporter, and a higher expression of OGDH in rat astrocytes versus neuroblastoma N2A. According to the data, the astrocytic OGDH may be up-regulated by an increase in intracellular ThDP, while the neuroblastomal OGDH functions at full ThDP saturation. Indeed, in rat astrocytes and brain cortex, OGDH inhibition by succinyl phosphonate (SP) enlarged the pool of thiamine compounds. Increased ThDP level in response to the OGDH inhibition presumably up-regulated the enzyme to compensate for a decrease in reducing power which occurred in SP-treated astrocytes. Under the same SP treatment of N2A cells, their thiamine pool and reducing power were unchanged, although SP action was evident from accumulation of glutamate. The presented data indicate that functional interplay between OGDH, other proteins of the tricarbocylic acid cycle and proteins of thiamine metabolism is an important determinant of physiology-specific networks and their homeostatic mechanisms.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias/efectos de los fármacos , Tiamina/metabolismo , Animales , Corteza Cerebral/metabolismo , Citoplasma/metabolismo , Ácido Glutámico/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Ratones , Mitocondrias/metabolismo , Organofosfonatos/metabolismo , Organofosfonatos/farmacología , Succinatos/metabolismo , Succinatos/farmacología , Tiamina Pirofosfato/metabolismoRESUMEN
2-Oxo acid dehydrogenase complexes are important metabolic checkpoints functioning at the intercept of sugar and amino acid degradation. This review presents a short summary of architectural, catalytic, and regulatory principles of the complexes structure and function, based on recent advances in studies of well-characterized family members. Special attention is given to use of synthetic phosphonate and phosphinate analogs of 2-oxo acids as selective and efficient inhibitors of the cognate complexes in biological systems of bacterial, plant, and animal origin. We summarize our own results concerning the application of synthetic analogs of 2-oxo acids in situ and in vivo to reveal functional interactions between 2-oxo acid dehydrogenase complexes and other components of metabolic networks specific to different cells and tissues. Based on our study of glutamate excitotoxicity in cultured neurons, we show how a modulation of metabolism by specific inhibition of its key reaction may be employed to correct pathologies. This approach is further developed in our study on the action of the phosphonate analog of 2-oxoglutarate in animals. The study revealed that upregulation of 2-oxoglutarate dehydrogenase complex is involved in animal stress response and may provide increased resistance to damaging effects, underlying so-called preconditioning. The presented analysis of published data suggests synthetic inhibitors of metabolic checkpoints as promising tools to solve modern challenges of systems biology, metabolic engineering, and medicine.
Asunto(s)
Inhibidores Enzimáticos/química , Complejo Cetoglutarato Deshidrogenasa/química , Ácidos Cetoglutáricos/química , Organofosfonatos/química , Ácidos Fosfínicos/química , Animales , Humanos , Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Complejo Cetoglutarato Deshidrogenasa/fisiología , Cinética , Mitocondrias/enzimologíaRESUMEN
Neurodegenerative diseases are accompanied by reduced activity of mitochondrial α-ketoglutarate dehydrogenase multienzyme complex (KGDHC). We present a new cellular model to study molecular mechanisms of this association. By application of the highly specific and efficient inhibitor of KGDHC, succinyl phosphonate (SP), to cultured neurons, we characterized the concentration- and time-dependent consequences of decreased KGDHC activity for neuronal metabolism and viability. Metabolic profiling of SP-treated neurons established accumulation of α-ketoglutarate and pyruvate as indicators of the KGDHC inhibition and ensuing impairment of pyruvate oxidation in the tricarboxylic acid cycle. Concomitant increases in alanine, glutamate and γ-aminobutyrate indicated a scavenging of the accumulated pyruvate and α-ketoglutarate by transamination and further decarboxylation of glutamate. Changes among other amino acids were in accordance with their potential to react with α-ketoglutarate or products of its transamination and serve as fuel compensating for the KGDHC block. Disturbances in neuronal amino acid pool were accompanied by changed polyamines, decreased total protein and increased thymine, suggesting increased catabolism of amino acids to decrease translation and affect DNA turnover/repair. The ensuing ATP salvage was observed as the paradoxical increase in neuronal ATP by mitochondrial inhibitor SP. Extensive exposure of neurons to SP reduced viability, as revealed by both the ATP- and NAD(P)H-dependent viability tests. Thus, we provide experimental evidence on the KGDHC impairment as a cause of neurodegeneration and decipher underlying molecular mechanisms, exposing the key regulatory complex of the tricarboxylic acid cycle as a promising target for directed regulation of neuronal function and survival.
Asunto(s)
Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Enfermedades Neurodegenerativas/enzimología , Neuronas/metabolismo , Animales , Carbohidratos/química , Células Cultivadas , Ciclo del Ácido Cítrico/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Metaboloma/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Enfermedades Neurodegenerativas/patología , Neuronas/citología , Neuronas/efectos de los fármacos , Organofosfonatos/química , Organofosfonatos/farmacología , Oxidación-Reducción , Ratas , Succinatos/química , Succinatos/farmacologíaRESUMEN
Decreased activity of the mitochondrial 2-oxoglutarate dehydrogenase complex (OGDHC) in brain accompanies neurodegenerative diseases. To reveal molecular mechanisms of this association, we treated rats with a specific inhibitor of OGDHC, succinyl phosphonate, or exposed them to hypoxic stress. In males treated with succinyl phosphonate and in pregnancy-sensitized females experiencing acute hypobaric hypoxia, we revealed upregulation of brain OGDHC (within 24 hours), with the activity increase presumably representing the compensatory response of brain to the OGDHC inhibition. This up-regulation of brain OGDHC was accompanied by an increase in exploratory activity and a decrease in anxiety of the experimental animals. Remarkably, the hypoxia-induced elevation of brain OGDHC and most of the associated behavioral changes were abrogated by succinyl phosphonate. The antagonistic action of hypoxia and succinyl phosphonate demonstrates potential therapeutic significance of the OGDHC regulation by the phosphonate analogs of 2-oxoglutarate.
RESUMEN
The alpha-ketoglutarate dehydrogenase complex (KGDHC) which catalyzes the conversion of alpha-ketoglutarate to succinyl-CoA and NADH in mitochondria, is known to generate O(2).- in vitro. To find out if KGDHC contributes to neuronal reactive oxygen species (ROS) increase in situ, we investigated whether the specific inhibitors of cellular KGDHC, succinyl phosphonate (SP) and the SP triethyl ester (TESP), might affect the glutamate-induced ROS production in cultured hippocampal neurons from rats. The concentration-dependent decrease in the mitochondrial potential of the glutamate-overstimulated neurons in the presence of SP or TESP indicated that under the conditions inducing neuronal ROS generation, the inhibitors are delivered to mitochondria, and their subsequent inhibition of KGDHC decreases the mitochondrial potential. The production of O(2).- was detected by reaction with hydroethidine. The distribution of the resulting fluorescence of DNA-ethidium coincided with that of the mitochondrial marker Mitotracker, pointing to the mitochondrial origin of the hydroethidine-detected ROS in response to glutamate (100 microM). At 200 microM, both TESP and SP administered together with glutamate, inhibited the glutamate-induced ROS production by about 20%, with the inhibition increasing to 44% at 500 microM TESP. The decrease in neuronal ROS by specific inhibitors of KGDHC demonstrates that KGDHC is a source of ROS in cultured neurons responding to glutamate. However, increasing the concentration of the strongest KGDHC inhibitor SP to 500 microM even increased the ROS production compared with glutamate alone, presumably due to secondary effects arising upon the strong KGDHC inhibition. Our work extends the current understanding of the glutamate-induced ROS generation in neurons, shedding light on the pathological mechanisms of the KGDHC involvement in glutamate neurotoxicity. In conclusion, potent KGDHC inhibitors are promising diagnostic tools for in situ study of neurodegenerative mechanisms.
Asunto(s)
Hipocampo/citología , Complejo Cetoglutarato Deshidrogenasa/fisiología , Neuronas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Mezclas Complejas/farmacología , ADN , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Etidio/análogos & derivados , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Organofosfonatos/farmacología , Ratas , Succinatos/farmacología , Factores de TiempoRESUMEN
The 2-oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2-oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nM, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2-oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mM, respectively. The system has a high specificity for 2-oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mM substrate inhibition is observed. From steady-state inhibition experiments with substrate analogs, two substrate-binding modes are revealed at different degrees of saturation of the enzyme with 2-oxoglutarate. At low substrate concentrations (10(-6) to 10(-5) M), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10(-4) to 10(-3) M) the relative contribution of the 2-oxo group in the binding increases. A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2-oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site. The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.
Asunto(s)
Azotobacter vinelandii/enzimología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Aciltransferasas/aislamiento & purificación , Aciltransferasas/metabolismo , Sitios de Unión , Dihidrolipoamida Deshidrogenasa/aislamiento & purificación , Dihidrolipoamida Deshidrogenasa/metabolismo , Flavinas/química , Complejo Cetoglutarato Deshidrogenasa/química , Complejo Cetoglutarato Deshidrogenasa/aislamiento & purificación , Ácidos Cetoglutáricos/metabolismo , Cinética , Magnesio/metabolismo , Ácido Oxaloacético/metabolismo , Tiamina Pirofosfato/químicaRESUMEN
Many halogenated foreign compounds are detoxified by conversion to the corresponding cysteine S-conjugate, which is N-acetylated and excreted. However, several halogenated cysteine S-conjugates [e.g. S-(1,1,2,2-tetrafluoroethy)-L-cysteine (TFEC)] are converted to mitochondrial toxicants by cysteine S-conjugate beta-lyases. In the present work, we showed that TFEC appreciably inactivated highly purified alpha-ketoglutarate dehydrogenase complex (KGDHC) in the presence of a cysteine S-conjugate beta-lyase. Incubation of PC12 cells (which contain endogenous cysteine S-conjugate beta-lyase activity) with TFEC led to a concentration- and time-dependent loss of endogenous KGDHC activity. A 24-hr exposure to 1 mM TFEC decreased KGDHC activity in the cells by 90%. Although treatment with TFEC did not inhibit intrinsic pyruvate dehydrogenase complex (PDHC) activity, it inhibited dichloroacetate/Mg2+-mediated activation/dephosphorylation of PDHC in the PC12 cells by 90%. To determine the selectivity of enzymes targeted by TFEC, several cytosolic and mitochondrial enzymes involved in energy metabolism [malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, cytosolic and mitochondrial aspartate aminotransferases (AspAT)] were also assayed in the PC12 cells exposed to 1 mM TFEC for 24 hr. Of these enzymes, only mitochondrial AspAT, a key enzyme of the malate-aspartate shuttle, was inhibited. The present results demonstrate a selective vulnerability of mitochondrial enzymes to toxic cysteine S-conjugates. The data indicate that TFEC may be a useful cellular/mitochondrial toxicant for elucidating the consequences of the diminished mitochondrial function that accompanies numerous neurodegenerative diseases.
Asunto(s)
Cisteína/análogos & derivados , Inhibidores Enzimáticos/farmacología , Hidrocarburos Fluorados/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Animales , Liasas de Carbono-Azufre/metabolismo , Cisteína/farmacología , Metabolismo Energético/efectos de los fármacos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias Cardíacas/enzimología , Células PC12 , Complejo Piruvato Deshidrogenasa/metabolismo , Ratas , Transaminasas/metabolismoRESUMEN
The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.
Asunto(s)
Cetona Oxidorreductasas/metabolismo , Complejos Multienzimáticos/metabolismo , Tiorredoxinas/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad Estática , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
Selective tryptic proteolysis of the mammalian alpha-ketoglutarate dehydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its alpha-ketoglutarate dehydrogenase (E1) component, which promotes the dissociation of the dihydrolipoamide dehydrogenase (E3) enzyme and also a fully active E1' fragment. Similarities between the N-terminal region of E1 and the dihydrolipoamide acetyltransferase (E2) and E3-binding components (E3BP) of the pyruvate dehydrogenase complex are highlighted by the specific cross-reactivities of subunit-specific antisera. Analysis of the pattern of release of E1 and E1' polypeptides from the OGDC during tryptic inactivation suggests that both polypeptide chains of individual E1 homodimers must be cleaved to permit the dissociation of the E1 and E3 components. A new protocol has been devised that promotes E1 dissociation from the oligomeric dihydrolipoamide succinyltransferase (E2) core in an active state. Significant levels of overall OGDC reconstitution could also be achieved by re-mixing the constituent enzymes in stoichiometric amounts. Moreover, a high affinity interaction has been demonstrated between the homodimeric E1 and E3 components, which form a stable subcomplex comprising single copies of these two enzymes.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/química , Complejo Cetoglutarato Deshidrogenasa/química , Conformación Proteica , Animales , Bovinos , Cromatografía en Gel , Dihidrolipoamida Deshidrogenasa/aislamiento & purificación , Dihidrolipoamida Deshidrogenasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Complejo Cetoglutarato Deshidrogenasa/aislamiento & purificación , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Cinética , Sustancias Macromoleculares , Cloruro de Magnesio/farmacología , Mamíferos , Peso Molecular , Miocardio/enzimologíaRESUMEN
Disulfide containing proteins--thioredoxins from E. coli and pig heart mitochondria--were characterized by sodium dodecyl sulfate (SDS)-electrophoresis and high performance capillary electrophoresis (HPCE). Following the mitochondrial thioredoxin samples at different stages of purification, we found that their electrophoretic patterns vary, dependent on the redox condition of isolation, preparation of the samples for SDS-electrophoresis, and sample storage. All these factors influenced the relative intensities of several protein bands with thioredoxin-like mobility, whereas the sample storage also resulted in the appearance of SDS- and dithiothreitol (DTT)-resistant high molecular mass forms, probably thioredoxin dimers. The multiple forms of the thioredoxin from pig heart mitochondria in SDS-electrophoresis might be dependent on the oxidation state of the protein cysteine residues. A commercial preparation of the thioredoxin from E. coli did not exhibit any changes in mobility in SDS gels whether the sample was prepared with or without DTT. After the final purification step no correlation was found between mitochondrial thioredoxin activity, determined in the insulin assay, and its purity in SDS-electrophoresis. A correlation was, however, found when analyzing the thioredoxin by HPCE. The latter approach demonstrated the heterogeneity of the thioredoxin samples homogeneous on SDS electrophoresis, only one of the several HPCE peaks being active in the insulin assay. Also, thioredoxin from E. coli, homogeneous on SDS-electrophoresis, was found heterogeneous on HPCE. The peak corresponding to the insulin-dependent thioredoxin activity was split into two by DTT treatment, suggesting that redox transformations of thioredoxin could be followed by HPCE.
Asunto(s)
Proteínas Bacterianas/química , Electroforesis Capilar/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Tiorredoxinas/química , Animales , Escherichia coli , Dodecil Sulfato de Sodio , PorcinosRESUMEN
The regulation of mitochondrial dehydrogenases of 2-oxoacids by thioredoxin is established. It is found that at low NAD+ and saturating concentrations of 2-oxoacids and CoA, inactivation of 2-oxoacid dehydrogenase complexes takes place, preventing NAD+ reduction under such conditions. However, addition of oxidized E. coli thioredoxin to the reaction medium without dithiothreitol allows effective NAD+ reduction at this substrate ratio. Product accumulation curves show that thioredoxin activates the complexes by protecting them from the inactivation observed in the conditions when the complex-bound dihydrolipoate is accumulated. Disappearance of the activatory effect of thioredoxin after its treatment with SH-specific reagents indicates the involvement of the redox-active cysteine couple of thioredoxin in its activation of 2-oxoacid dehydrogenase complexes. The redox-inactive thioredoxin not only shows no activation, but in fact exerts an inhibitory effect. The inhibition manifests the complex formation between SH-modified thioredoxin and dehydrogenase systems, involving amino acid residues of thioredoxin other than cysteine. High efficiency of thioredoxin from E. coli as compared to chloroplast thioredoxin f and glutathione disulfide is revealed. This indicates the importance of specific protein structure also for the influence of the redox-active thioredoxin upon the 2-oxoacid dehydrogenase complexes. The results obtained suggest that these key enzyme systems of mitochondrial metabolism represent previously unidentified targets for the action of mitochondrial thioredoxin, which is known to resemble the E. coli counterpart studies in this work.
Asunto(s)
Cetona Oxidorreductasas/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Tiorredoxinas/farmacología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Animales , Cisteína/análisis , Activación Enzimática/efectos de los fármacos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias Cardíacas/enzimología , NAD , Complejo Piruvato Deshidrogenasa/metabolismo , Reactivos de Sulfhidrilo , PorcinosRESUMEN
Inhibition of alpha-ketoglutarate dehydrogenase (KGD) by dicarboxylates with (oxaloacetate and ketomalonate) and without (malonate, succinate, and glutarate) alpha-keto group was studied. Ketodicarboxylates at low concentrations inhibit KGD in competitive manner. Increase in their concentrations results in appearance of the noncompetitive component. The extent of KGD inhibition by keto dicarboxylates increases with structural similarity of the inhibitor and the substrate, irrespective of preliminary incubation of the enzyme with the inhibitor. This is indicative of blocking the substrate-binding site of KGD by dicarboxylates. In contrast, inhibitory effect of dicarboxylates which contain no keto group increases as their structural similarity with the substrate decreases. Saturation of KGD with dicarboxylates of this type does not completely suppress the enzymatic activity. Alternatively, these analogs display competitive mode of inhibition. Analysis of the data obtained suggests that these dicarboxylates produce catalytically active triple complex keto substrate-KGD-dicarboxylate and that KGD which enters the composition of such a complex inhibits a decreased affinity for the keto substrate as a result of the inhibitor binding.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Ácidos Cetoglutáricos/química , Músculo Esquelético/enzimología , Animales , Catálisis , Columbidae , Ácidos Cetoglutáricos/farmacología , Cinética , Malonatos/farmacología , Mitocondrias Musculares/metabolismo , Oxaloacetatos/farmacología , Ácido Succínico/farmacologíaRESUMEN
The activity of some muscle alpha-ketoglutarate dehydrogenase complexes (KGDC) decreases during their enzymatic reaction as a result of inactivation of the first component of the complex, namely, alpha-ketoglutarate dehydrogenase (KGD). This decrease is associated with transformation of the complex produced by KGD and alpha-keto substrate in the course of oxidative phosphorylation. Kinetics of KGD inactivation during the reaction depends on teh keto substrate structure. When alpha-ketoglutarate (KG) is used as a substrate, KGD inactivation occurs in two stages. The major (irreversible) decrease in its activity is observed during the first minutes of reaction. During reaction with a catalytically active KG analog, alpha-ketoadipate (KA), the enzyme is irreversibly inactivated in one stage. This suggests that the reversible stage of inactivation is due to the high rate of catalysis, which is characteristic of KG-utilizing reactions. Decrease in the rate of KG oxidation after treatment of the enzyme with sulfhydryl reagents eliminates this reversible stage. Preincubation of KGD with KG phospho-derivatives (succinylphosphonate or its monomethyl ether) changes the properties of KGD in a similar way to the reversible decrease of activity during catalysis. The arginine residue of KGD, which is essential for enzymatic activity, becomes inaccessible for butanedione in the complexes of KGD with some phospho-derivatives. The data suggest that the reversible inactivation of KGD in the course of catalysis is due to an interaction of the leaving substrate carboxyl with the essential arginine residue of the enzyme.