RESUMEN

The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.

Asunto(s)

Genómica , Metabolómica , Neoplasias/patología , Urea/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Animales , Aspartato Carbamoiltransferasa/genética , Aspartato Carbamoiltransferasa/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Línea Celular Tumoral , Dihidroorotasa/genética , Dihidroorotasa/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Proteínas de Transporte de Membrana Mitocondrial , Neoplasias/metabolismo , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Fosforilación/efectos de los fármacos , Pirimidinas/biosíntesis , Pirimidinas/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Transcarbamylases reversibly transfer a carbamyl group from carbamylphosphate (CP) to an amine. Although aspartate transcarbamylase and ornithine transcarbamylase (OTC) are well characterized, little was known about putrescine transcarbamylase (PTC), the enzyme that generates CP for ATP production in the fermentative catabolism of agmatine. We demonstrate that PTC (from Enterococcus faecalis), in addition to using putrescine, can utilize L-ornithine as a poor substrate. Crystal structures at 2.5 Šand 2.0 Šresolutions of PTC bound to its respective bisubstrate analog inhibitors for putrescine and ornithine use, N-(phosphonoacetyl)-putrescine and δ-N-(phosphonoacetyl)-L-ornithine, shed light on PTC preference for putrescine. Except for a highly prominent C-terminal helix that projects away and embraces an adjacent subunit, PTC closely resembles OTCs, suggesting recent divergence of the two enzymes. Since differences between the respective 230 and SMG loops of PTC and OTC appeared to account for the differential preference of these enzymes for putrescine and ornithine, we engineered the 230-loop of PTC to make it to resemble the SMG loop of OTCs, increasing the activity with ornithine and greatly decreasing the activity with putrescine. We also examined the role of the C-terminal helix that appears a constant and exclusive PTC trait. The enzyme lacking this helix remained active but the PTC trimer stability appeared decreased, since some of the enzyme eluted as monomers from a gel filtration column. In addition, truncated PTC tended to aggregate to hexamers, as shown both chromatographically and by X-ray crystallography. Therefore, the extra C-terminal helix plays a dual role: it stabilizes the PTC trimer and, by shielding helix 1 of an adjacent subunit, it prevents the supratrimeric oligomerizations of obscure significance observed with some OTCs. Guided by the structural data we identify signature traits that permit easy and unambiguous annotation of PTC sequences.

Asunto(s)

Agmatina/metabolismo , Transferasas de Carboxilo y Carbamoilo/química , Transferasas de Carboxilo y Carbamoilo/metabolismo , Fermentación , Familia de Multigenes , Agmatina/química , Secuencia de Aminoácidos , Biocatálisis , Transferasas de Carboxilo y Carbamoilo/antagonistas & inhibidores , Dominio Catalítico , Cristalografía por Rayos X , Enterococcus faecalis/enzimología , Hidrolasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Ornitina/química , Ornitina/metabolismo , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ingeniería de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Putrescina/química , Putrescina/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato

RESUMEN

A scalable four-step synthesis of the ornithine transcarbamylase inhibitor N(5)-phosphonoacetyl-l-ornithine (PALO) is achieved through boroxazolidinone protection of ornithine. Investigations in the model organism Saccharomyces cerevisiae found that, in contrast to a previous report, PALO did not influence growth rate or expression of genes involved in arginine metabolism.

Asunto(s)

Arginina/metabolismo , Ornitina/análogos & derivados , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulación de la Expresión Génica , Ornitina/síntesis química , Ornitina/química , Ornitina/farmacología , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética

RESUMEN

(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).

Asunto(s)

Farmacorresistencia Bacteriana , Ornitina Carbamoiltransferasa/aislamiento & purificación , Ornitina Carbamoiltransferasa/metabolismo , Ornitina/análogos & derivados , Ornitina/farmacología , Pseudomonas/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Medición de Intercambio de Deuterio , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Cinética , Modelos Químicos , Datos de Secuencia Molecular , Ornitina/química , Ornitina/metabolismo , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/biosíntesis , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato

RESUMEN

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25 degrees C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase(Mab) displays higher K(m) values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO). OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K(m) values and the lower sensitivity to PALO. The K(m) for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k(cat)/K(m)) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K(m) value for ornithine observed when the temperature is brought from 20 to 5 degrees C. OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.

Asunto(s)

Moritella/fisiología , Ornitina Carbamoiltransferasa/química , Ornitina Carbamoiltransferasa/metabolismo , Ornitina/análogos & derivados , Adaptación Biológica , Secuencias de Aminoácidos , Frío , Activación Enzimática , Estabilidad de Enzimas , Cinética , Mutación , Ornitina/metabolismo , Ornitina/farmacología , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/genética , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia , Temperatura

RESUMEN

The crystal structure is reported at 1.8 A resolution of Escherichia coli ornithine transcarbamoylase in complex with the active derivative of phaseolotoxin from Pseudomonas syringae pv. phaseolicola, N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine. Electron density reveals that the complex is not a covalent adduct as previously thought. Kinetic data confirm that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine exhibits reversible inhibition with a half-life in the order of approximately 22 h and a dissociation constant of K(D) = 1.6 x 10(-12) m at 37 degrees C and pH 8.0. Observed hydrogen bonding about the chiral tetrahedral phosphorus of the inhibitor is consistent only with the presence of the R enantiomer. A strong interaction is also observed between Arg(57) Nepsilon and the P-N-S bridging nitrogen indicating that imino tautomers of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine are present in the bound state. An imino tautomer of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine is structurally analogous to the proposed reaction transition state. Hence, we propose that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine, with its three unique N-P bonds, represents a true transition state analogue for ornithine transcarbamoylases, consistent with the tight binding kinetics observed.

Asunto(s)

Ornitina Carbamoiltransferasa/metabolismo , Ornitina/análogos & derivados , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Electrones , Escherichia coli/enzimología , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Ornitina/química , Ornitina/farmacología , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/química , Conformación Proteica , Factores de Tiempo

RESUMEN

The allosteric catabolic ornithine carbamoyltransferase (OTCase) from Pseudomonas aeruginosa, a dodecamer build up of four trimers of identical subunits, shows strong carbamoylphosphate homotropic co-operativity. Its activity is allosterically inhibited by spermidine and activated by AMP. Modified forms of the enzyme exhibiting substantial alterations in both homotropic and heterotropic interactions were recently obtained. We report here the first detailed kinetic characterization of homotropic and heterotropic modulations in allosteric wild-type and in engineered OTCases. Homotropic co-operativity for the saturation either by citrulline or arsenate was also observed when arsenate was utilised as an alternate substrate of the reverse reaction. Amino acid substitution of glutamate 105 by a glycine produces an enzyme devoid of homotropic interactions between the catalytic sites for carbamoylphosphate. This mutant, which is blocked in an active conformation, is still sensitive to the allosteric effector AMP, which increases affinity with respect to the substrate, carbamoylphosphate. It is also observed that homotropic co-operative interactions do not reappear in the E105G enzyme upon strong inhibition by the allosteric inhibitor of the wild-type enzyme, spermidine.Replacement of residues 34 to 101 of the native enzyme by the homologous amino acids of anabolic Escherichia coli OTCase produces a trimeric enzyme which retains reduced homotropic co-operativity. Activation by AMP and inhibition by spermidine of this chimaeric OTCase do not affect carbamoylphosphate homotropic co-operativity. AMP acts by reducing the concentration of substrate at half maximum velocity while spermidine acts in the inverse way. These observations indicate that in the two mutant forms of OTCase, homotropic and heterotropic interactions can be uncoupled and therefore must involve different molecular mechanisms. Furthermore, the results of stimulation of enzyme activity by phosphate, arsenate, pyrophosphate and phosphonoacetyl-l-ornithine on wild-type and mutant OTCases suggest that the physiological substrate phosphate, besides acting at the catalytic site, may act at an allosteric site. On the other hand, pyrophosphate and phosphonoacetyl-l-ornithine activation results exclusively from interactions of this effector with the active site residues.

Asunto(s)

Proteínas de Escherichia coli , Ornitina Carbamoiltransferasa/metabolismo , Proteínas Quinasas , Pseudomonas aeruginosa/enzimología , Adenosina Monofosfato/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Arseniatos/farmacología , Proteínas Bacterianas/química , Unión Competitiva , Difosfatos/metabolismo , Activación Enzimática , Represión Enzimática , Cinética , Proteínas de la Membrana/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Ornitina/análogos & derivados , Ornitina/farmacología , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/genética , Fosfatos/metabolismo , Pseudomonas aeruginosa/metabolismo , Homología de Secuencia de Aminoácido , Espermidina/farmacología

RESUMEN

A cluster of genes involved in the production of phaseolotoxin, a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, contains eight (phtA through phtH) complementation groups (Y. X. Zhang, K. B. Rowley, and S. S. Patil, J. Bacteriol., 175:6451-6458, 1993). In this study, sequencing of the region encompassing the phtE locus revealed six putative open reading frames (ORFs), each preceded by a putative ribosomal binding site, and all oriented in the same direction. Reverse transcription-polymerase chain reaction suggested that the phtE locus is transcribed as one large (6.4 kb) transcript, indicating that the ORFs constitute an operon. Primer extension analysis showed that the transcript begins at a T, located 31 bp upstream of the ATG codon of ORF1. Comparison of the sequences of the putative ORFs with the sequences of known genes revealed that ORF3, encoding a protein containing 395 amino acids, has 55% similarity to the acetylornithine aminotransferase gene from Escherichia coli, and the ornithine aminotransferase genes from other organisms. A lysine residue that is a binding site for pyridoxal phosphate and an arginine residue that is a binding site for the alpha-carboxylate group of the substrate are conserved in ORF3. These data suggest that ORF3 encodes a protein involved in the biosynthesis of ornithine, a constituent of phaseolotoxin. ORF5, encoding a peptide of 378 amino acid residues, possesses a helix-turn-helix motif at the C-terminal end that is characteristic of the AraC family of transcriptional factors, and there is a possible leucine zipper at the N-terminal end of this peptide. ORF6, encoding a protein of 327 amino acids, has about 40% similarity with the fatty acid desaturase gene, desA, of Synechocystis Pcc6803 and considerable similarity with fatty acid desaturase genes from other organisms. ORF6 and desA show very similar hydropathy profiles and both contain a copper binding signature. Computer searches did not discover significant homologies in the data base for the other ORFs, but hydropathy analysis showed that all of them contain one to several hydrophobic domains, suggesting that the gene products of these ORFs may be membrane associated.

Asunto(s)

Proteínas Bacterianas , Toxinas Bacterianas/biosíntesis , Exotoxinas/biosíntesis , Genes Bacterianos , Familia de Multigenes , Pseudomonas/genética , Factores de Transcripción , Secuencia de Aminoácidos , Factor de Transcripción de AraC , Secuencia de Bases , Inhibidores Enzimáticos , Proteínas de Escherichia coli , Ácido Graso Desaturasas/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Ornitina/análogos & derivados , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Péptido Sintasas/genética , Proteínas Represoras/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transaminasas/genética

RESUMEN

The ornithine carbamoyltransferases (OTCases) from the beta-lactam-producing actinomycetes Streptomyces clavuligerus and Nocardia lactamdurans have been purified to near-homogeneity by delta-N-phosphonoacetylornithine-Sepharose 4B affinity chromatography. The S. clavuligerus and N. lactamdurans OTCases monomers had a molecular mass of 37 kDa. The native OTCases of S. clavuligerus, N. lactamdurans and Streptomyces coelicolor had molecular masses of 248, 251 and 247 kDa respectively, which correspond to a hexameric structure. The apparent K(m) values for ornithine and carbamoylphosphate of the S. clavuligerus enzyme were respectively 2.3 and 6.0 mM at pH 8.0. The enzyme showed a reverse activity on citrulline and used lysine and putrescine as substrates. The hexameric complex showed coupled arginase-OTCase activities and was able to convert arginine into citrulline in a carbamoylphosphate-dependent manner. The requirement for carbamoylphosphate might prevent the arginase-OTCase complex from carrying out a futile cycle of arginine biosynthesis and degradation.

Asunto(s)

Arginasa/metabolismo , Cefamicinas/biosíntesis , Nocardia/enzimología , Ornitina Carbamoiltransferasa/metabolismo , Streptomyces/enzimología , Regulación Alostérica , Arginina/metabolismo , Biopolímeros , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Cinética , Peso Molecular , Nocardia/metabolismo , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/aislamiento & purificación , Streptomyces/metabolismo , Especificidad por Sustrato

RESUMEN

Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.

Asunto(s)

Hígado/enzimología , Lisina , Ornitina Carbamoiltransferasa/química , Ornitina Carbamoiltransferasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Borohidruros/farmacología , Carbamoil Fosfato/farmacología , Delfines , Inhibidores Enzimáticos , Escherichia coli/enzimología , Humanos , Cinética , Datos de Secuencia Molecular , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Fosfato de Piridoxal/farmacología , Homología de Secuencia de Aminoácido , Tripsina

RESUMEN

Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]0.5) and high cooperativity toward this substrate. We have isolated mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa. Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphosphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.

Asunto(s)

Ornitina Carbamoiltransferasa/metabolismo , Pseudomonas aeruginosa/enzimología , Regulación Alostérica , Animales , Activación Enzimática/genética , Metionina/química , Mutación , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/genética , Plásmidos , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/genética

RESUMEN

The probable involvement of hepatic carbamyl-P in the reciprocal relationship between hepatic ureagenesis and glycogenesis from glucose was explored. Isolated perfused liver preparations from 48-h fasted rats were employed. Moderate (9.2 mM) and relatively high levels of glucose (34 mM) were perfused. Hepatic glycogenesis, glucose-6-P, carbamyl-P, and citrulline levels, hepatic urea formation, and ureagenesis based upon perfusate urea levels were measured. Experimental probes selected to modify hepatic ureagenesis and carbamyl-P production and utilization included: (a) NH4Cl, maintained at 5 mM by continuous infusion (NH4+ is a substrate for carbamyl-P synthase I and glutamate dehydrogenase); (b) norvaline, an inhibitor of ornithine transcarbamylase which catalyzes the first committed step in the urea cycle; and (c) ethoxyzolamide, an inhibitor of carbonic anhydrase which produces HCO3-, an essential substrate for carbamyl-P synthase I. NH4+ increased ureagenesis and decreased glycogenesis. The inclusion of norvaline with NH4+ decreased ureagenesis and increased glycogenesis. Ethoxyzolamide with or without NH4+ inhibited both ureagenesis and glycogenesis, and decreased the hepatic glucose-6-P level. Glycogenesis was greater at 34 mM than 9.2 mM glucose, increased in norvaline-containing preparations correlative with increased availability of carbamyl-P, and decreased when carbamyl-P formation was inhibited by ethoxyzolamide. Kinetic analysis indicated a Km, Glc of 31 mM for glucose phosphorylation preliminary to glycogenesis. Glycogen formation via the "indirect pathway" (i.e. involving extrahepatic glycolysis, transport of lactate to the liver, and glyconeogenesis therefrom) was quantitatively insufficient to account for the observed glycogenesis. Glucokinase is contraindicated by the inverse relationship between hepatic glycogenesis and ATP availability in the ethoxyzolamide-treated preparations. In contrast, carbamyl-P:glucose phosphotransferase activity of the glucose-6-phosphatase system has the characteristics to bridge hepatic ureagenesis and glycogenesis.

Asunto(s)

Cloruro de Amonio/farmacología , Etoxzolamida/farmacología , Glucosa/metabolismo , Glucógeno Hepático/biosíntesis , Hígado/metabolismo , Urea/metabolismo , Valina/análogos & derivados , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Inhibidores de Anhidrasa Carbónica/farmacología , Glucoquinasa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Hexoquinasa/metabolismo , Técnicas In Vitro , Cinética , Hígado/efectos de los fármacos , Modelos Biológicos , Tamaño de los Órganos , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Perfusión , Ratas , Valina/farmacología

RESUMEN

In crude protein extracts of aseptically grown plantlets of Datura innoxia ornithine carbamoyltransferase was detected. The ornithine carbamoyltransferase of plant extracts was inhibited by phaseolotoxin. The level of this inhibition was dependent on the concentration of the toxin applied. Subsequent accumulation of ornithine in plant vegetative parts was observed. Phaseolotoxin influenced both the vitability and alkaloid accumulation of treated plants.

Asunto(s)

Alcaloides/metabolismo , Exotoxinas/farmacología , Ornitina Carbamoiltransferasa/metabolismo , Plantas/metabolismo , Alcaloides/aislamiento & purificación , Ornitina/análogos & derivados , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Plantas/efectos de los fármacos , Plantas/enzimología

RESUMEN

Phaseolotoxin [N delta(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl- homoarginine] produced by Pseudomonas syringae pv. phaseolicola, the bean halo blight pathogen, is a potent inhibitor of ornithine carbamoyltransferase (OCT). Inhibition of OCT in infected plants leads to chlorosis and growth inhibition. A genomic cosmid clone, pHK120, containing a 25-kb fragment of DNA from a wild-type strain of P. syringae pv. phaseolicola restores toxin production in Tox- mutants. Tn5 mutagenesis of pHK120 and marker exchange of pHK120::Tn5 plasmids in the wild-type strain resulted in the isolation of 39 chromosomal mutants that harbor Tn5 insertions at known positions. Toxin bioassays revealed that 28 of the mutants, with Tn5 insertions distributed throughout the insert of pHK120, were Tox-, indicating that a functional locus for toxin production in each mutant was inactivated. Complementation analysis was done by testing for toxin production strains that carried a genomic Tn5 at one location and a plasmid-borne Tn5 at another location (pair complementation). Pair complementation analysis of nine marker exchange mutants and a random genomic Tn5 mutant revealed that there are a minimum of eight toxin loci (phtA through phtH) in pHK120. Mutants carrying Tn5 insertions in the phtA, phtD, and phtF loci were complemented by deletion subclones containing fragments from pHK120; mutants carrying Tn5 insertions in the phtC locus were partially complemented by a subclone, and mutants carrying Tn5 insertions in the phtB, phtE, phtG, and phtH loci were not complemented by any of the available subclones. A comparison of the insert from pHK120 with that from pRCP17, a clone reported previously (R. C. Peet, P. B. Lindgren, D. K. Wills, and N. J. Panopoulos, J. Bacteriol. 166:1096-1105, 1986) by another laboratory to contain some of the phaseolotoxin genes and the phaseolotoxin-resistant OCT gene, revealed that the inserts in these two cosmids overlap but differ in important respects.

Asunto(s)

Toxinas Bacterianas/biosíntesis , Exotoxinas/genética , Genes Bacterianos , Pseudomonas/genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , Prueba de Complementación Genética , Familia de Multigenes , Mutagénesis Insercional , Ornitina/análogos & derivados , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Pseudomonas/patogenicidad , Mapeo Restrictivo , Temperatura

RESUMEN

Mitochondrial preornithine transcarbamylase (p-OTC) and premalate dehydrogenase (p-MDH) are the only two matrix-located preproteins so far identified for which the proteolytic processing in vitro requires the formation of genuine processing intermediates, i-OTC and i-MDH, respectively. To establish the processing of other preproteins during import with respect to the two-step processing of p-OTC and p-MDH, the chelators EDTA and 1,10-phenanthroline were used to study the import and processing of rat prechaperonin 60 (p-cpn60) and p-OTC by mitochondria from four cpn60-containing organs. We found no evidence for a secondary processing step in the maturation of p-cpn60, but a clear requirement for two-step processing of p-OTC, even in three organs which do not contain ornithine transcarbamylase. The metal-ion requirement of the p-OTC processing activities in the organelle is consistent with the proposition that the mitochondrial processing protease (MPP) and mitochondrial intermediate peptidase (MIP) activities defined in vitro [Kalousek, F., Hendrick, J.P. & Rosenberg, L. E. (1988) Proc. Natl Acad. Sci. USA 85, 7536-7540] are responsible for precursor processing in vivo. The authenticity of two-step processing in vivo was, furthermore, established by demonstrating that i-OTC accumulates to high levels in Spodoptora frugiperda insect cells supplemented with MnCl2. The inability of the insect cells to process p-OTC fully is not a characteristic of cells grown in culture since cultured rat hepatoma cells process p-OTC to the fully processed m-OTC. Finally, we find that the import and processing of p-cpn60 and p-OTC is inhibited in an identical fashion by presequence-bovine-serum-albumin conjugates. The differences in proteolytic maturation between p-cpn60 and p-OTC are therefore not likely to result from different import pathways as the two precursors compete for common components of the import apparatus.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitocondrias Hepáticas/enzimología , Ornitina Carbamoiltransferasa/metabolismo , Precursores de Proteínas/metabolismo , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Western Blotting , Chaperonina 60 , Ácido Edético/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Riñón/enzimología , Riñón/ultraestructura , Masculino , Mitocondrias/enzimología , Mitocondrias Hepáticas/efectos de los fármacos , Especificidad de Órganos , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Fenantrolinas/farmacología , Conejos , Ratas , Bazo/enzimología , Bazo/ultraestructura , Testículo/enzimología , Testículo/ultraestructura

RESUMEN

We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus. We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins. Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity. MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition. To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP. The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate. Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein. When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta. Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.

Asunto(s)

Endopeptidasas/metabolismo , Metaloendopeptidasas , Oligopéptidos/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Hígado/enzimología , Datos de Secuencia Molecular , NADH Deshidrogenasa/antagonistas & inhibidores , NADH Deshidrogenasa/metabolismo , Neurospora/enzimología , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/metabolismo , Procesamiento Proteico-Postraduccional , Ratas

RESUMEN

We have demonstrated that a synthetic peptide corresponding to the rat mitochondrial malate dehydrogenase (mMDH) transit peptide (TP-28) inhibits the binding of pre-mMDH to isolated mitochondria. Synthetic peptides derived from chloroplast transit peptide sequences, which have a similar net charge, did not inhibit import. In addition, this peptide (TP-28) inhibits import of ornithine transcarbamylase, another mitochondrial matrix protein, thus suggesting that common import pathways exist for both mMDH and ornithine transcarbamylase. A smaller synthetic peptide corresponding to residues 1-20 of the mMDH transit peptide (TP-20) also inhibits binding. However, several substitutions for leucine-13 in the smaller peptide relieve import inhibition, thus providing evidence that this neutral residue plays a crucial role in transit peptide binding to the mitochondrial surface. Proteolytic processing of pre-mMDH by a mitochondrial matrix fraction to both the mature and intermediate forms of mMDH was also inhibited by TP-28. The ability of synthetic peptides to inhibit distinct steps in the import of mitochondrial precursor proteins corresponds precisely to their ability to interact with the same components used by transit peptides on intact precursors. Furthermore, inhibition at multiple points along the import pathway reflects the functions of several independent structures contained within transit peptides.

Asunto(s)

Malato Deshidrogenasa/antagonistas & inhibidores , Mitocondrias Hepáticas/enzimología , Péptidos/farmacología , Precursores de Proteínas/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Hidrólisis , Malato Deshidrogenasa/metabolismo , Mitocondrias Hepáticas/metabolismo , Datos de Secuencia Molecular , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Péptidos/síntesis química , Precursores de Proteínas/metabolismo , Ratas

RESUMEN

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.

Asunto(s)

Mitocondrias Hepáticas/enzimología , Ornitina Carbamoiltransferasa/aislamiento & purificación , Animales , Carbamoil Fosfato/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cazón , Glutamina/metabolismo , Cinética , Peso Molecular , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Urea/biosíntesis , Valina/farmacología

RESUMEN

Ornithine carbamoyltransferase (OCTase) has been identified as a cadmium-binding protein in rat liver (Aoki et al., 1988), whose enzymatic activity is inhibited by cadmium and mercury ions. Cadmium is a competitive inhibitor of OCTase with respect to carbamoyl phosphate and a noncompetitive inhibitor with respect to ornithine. Data presented here show that in primary cultured rat liver parenchymal cells cadmium at concentrations of 7-20 microM inhibits urea production.

Asunto(s)

Cadmio/farmacología , Hígado/metabolismo , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Urea/biosíntesis , Animales , Técnicas In Vitro , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas

RESUMEN

Independently of their capacity to produce phytotoxins, strains of Pseudomonas syringae pv. phaseolicola contain two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) which differ in resistance to phaseolotoxin and Orn-P(O) (NH2)-NH-SO3 H (PNSOrn). At 18 degrees C, the optimal temperature for product formation, the balance of the two types of OCT was shifted in favour of the insensitive type in phaseolotoxin producing strains, and in favour of the sensitive one in strains with little or no toxin production. The results suggest a causal relationship between the existence of an insensitive enzyme and the synthesis of toxins.

Asunto(s)

Exotoxinas/biosíntesis , Ornitina Carbamoiltransferasa/metabolismo , Pseudomonas/metabolismo , Resistencia a Medicamentos , Exotoxinas/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Ornitina/análogos & derivados , Ornitina Carbamoiltransferasa/antagonistas & inhibidores