RESUMEN
Δ(1)-pyrroline-5-carboxylate (P5C) reductase (P5CR) catalyses the final step of proline synthesis in plants. In Arabidopsis thaliana, protein levels are correlated neither to the corresponding mRNA copy numbers, nor to intracellular proline concentrations. The occurrence of post-translational regulatory mechanisms has therefore been hypothesized, but never assessed. The purification of A. thaliana P5CR was achieved through either a six-step protocol from cultured cells, or heterologous expression of AtP5CR in Escherichia coli. The protein was characterized with respect to structural, kinetic, and biochemical properties. P5CR was able to use either NADPH or NADH as the electron donor, with contrasting affinities and maximum reaction rates. The presence of equimolar concentrations of NADP(+) completely suppressed the NADH-dependent activity, whereas the NADPH-dependent reaction was mildly affected. Proline inhibited only the NADH-dependent reaction. At physiological values, increasing concentrations of salt progressively inhibited the NADH-dependent activity, but were stimulatory of the NADPH-dependent reaction. The biochemical properties of A. thaliana P5CR suggest a complex regulation of enzyme activity by the redox status of the pyridine nucleotide pools, and the concentrations of proline and chloride in the cytosol. Data support a to date underestimated role of P5CR in controlling stress-induced proline accumulation.
Asunto(s)
Arabidopsis/enzimología , Cloruros/farmacología , Retroalimentación Fisiológica/efectos de los fármacos , NADP/metabolismo , NAD/metabolismo , Prolina/farmacología , Pirrolina Carboxilato Reductasas/antagonistas & inhibidores , Pirrolina Carboxilato Reductasas/metabolismo , Arabidopsis/efectos de los fármacos , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Iones , Oxidación-Reducción , Cloruro de Potasio/farmacología , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Cloruro de Sodio/farmacología , Especificidad por Sustrato/efectos de los fármacos , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
The gene SSO0495 (proC), which encodes pyrroline-5-carboxylate reductase (P5CR) from the thermoacidophilic archeon Sulfolobus solfataricus P2 (Ss-P5CR), was cloned and expressed. The purified recombinant enzyme catalyzes the thioproline dehydrogenase with concomitant oxidation of NAD(P)H to NAD(P)+. This archeal enzyme has an optimal alkaline pH in this reversible reaction and is thermostable with a half-life of approximately 30 min at 80 degrees C. At pH 9.0, the reverse activation rate is nearly 3-fold higher than at pH 7.0. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Ss-P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 degrees C. Diffraction data were obtained to a resolution of 3.5A and were suitable for X-ray structure determination.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/aislamiento & purificación , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Sulfolobus solfataricus/enzimología , Temperatura , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Cinética , NADP/química , NADP/metabolismo , Sulfolobus solfataricus/metabolismo , Tiazolidinas/química , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escherichia coli and purified to homogeneity by chromatography. Enzymatic assays of the wild-type protein were carried out using 3,4-dehydro-L-proline as substrate and NAD(+) as cofactor. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Human P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 degrees C. Diffraction data were obtained to a resolution of 2.8A and were suitable for high resolution X-ray structure determination.
Asunto(s)
Pirrolina Carboxilato Reductasas/aislamiento & purificación , Pirrolina Carboxilato Reductasas/metabolismo , Cristalización , Cristalografía por Rayos X , Expresión Génica , Humanos , Polímeros/química , Prolina/análogos & derivados , Prolina/metabolismo , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/genética , Solubilidad , AguaRESUMEN
Pyrroline-5-carboxylate reductase (P5CR) plays an important role in the survival of Mycobacterium tuberculosis and is related to virulence of this pathogen. RT-PCR analysis indicated that proC, encoding P5CR, was expressed at the transcriptional level cultured in vitro. The His-rMtP5CR with an N-terminal His-tag (His-rMtP5CR) was firstly purified in Escherichia coli and rMtP5CR was obtained by removal of the N-terminal fusion partner using enterokinase. His-rMtP5CR had considerable beta-pleated sheet analyzed by circular dichroism spectroscopy. The effect of pH, temperature, cations, denaturants, and detergents on the purified enzyme activity and stability was characterized. The N-terminal fusion partner was found to have very little effect on the biochemical properties of P5CR.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Pirrolina Carboxilato Reductasas/química , Células Cultivadas , Dicroismo Circular , Clonación Molecular , ADN/genética , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Cinética , Pirrolina Carboxilato Reductasas/genética , Pirrolina Carboxilato Reductasas/aislamiento & purificación , ARN/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , TemperaturaRESUMEN
L-proline is an amino acid that plays an important role in proteins uniquely contributing to protein folding, structure, and stability, and this amino acid serves as a sequence-recognition motif. Proline biosynthesis can occur via two pathways, one from glutamate and the other from arginine. In both pathways, the last step of biosynthesis, the conversion of delta1-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by delta1-pyrroline-5-carboxylate reductase (P5CR) using NAD(P)H as a cofactor. We have determined the first crystal structure of P5CR from two human pathogens, Neisseria meningitides and Streptococcus pyogenes, at 2.0 angstroms and 2.15 angstroms resolution, respectively. The catalytic unit of P5CR is a dimer composed of two domains, but the biological unit seems to be species-specific. The N-terminal domain of P5CR is an alpha/beta/alpha sandwich, a Rossmann fold. The C-terminal dimerization domain is rich in alpha-helices and shows domain swapping. Comparison of the native structure of P5CR to structures complexed with L-proline and NADP+ in two quite different primary sequence backgrounds provides unique information about key functional features: the active site and the catalytic mechanism. The inhibitory L-proline has been observed in the crystal structure.
Asunto(s)
Neisseria meningitidis/enzimología , Pirrolina Carboxilato Reductasas/química , Streptococcus pyogenes/enzimología , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Pirrolina Carboxilato Reductasas/metabolismo , Homología de Secuencia de Aminoácido , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
Delta(1)-Pyrroline-5-carboxylate reductase (P5CR) (EC 1.5.1.2. L-proline: NAD(P)-5-oxidoreductase), the second enzyme in the proline biosynthetic pathway, was purified from spinach (Spinacia oleracea L.) leaves. Following ammonium sulfate fractionation, purification was performed by several chromatographic methods: Blue Cellulofine, DEAE-TOYOPEARL, Sephacryl S-300 HR, and POROS QE/M. Two isoenzymes resolved by anion exchange chromatography were designated P5CR-1 and P5CR-2. Only P5CR-2 was purified from the intact chloroplasts, indicating differential distribution of the isoenzymes. P5CR isoenzymes, P5CR-1 and P5CR-2, are a homopolymer with an apparent molecular mass of 310 kDa, consisting of 10 to 12 subunits of about 28.5 kDa. P5CR-1 and P5CR-2 showed K(m) values of 9 and 19 microM for NADPH and values of 0.122 and 0.162 mM for Delta(1)-pyrroline-5-carboxylate (P5C), respectively. We decided partial amino acid sequences of P5CR-1 which showed the 70 to 80% homology to the deduced amino acid sequences of several plant P5CR cDNAs. Both isoenzymes had much lower affinity for NADH than for NADPH and were inhibited by free ATP and Mg(2+) ion. The inhibition was partially mitigated when ATP and Mg(2+) were added simultaneously to the reaction mixture. Cations at high concentration were inhibitory to P5CR activity. Interestingly, P5CR-2 was more stable to heat treatment at 40 degrees C than P5CR-1.
Asunto(s)
Pirrolina Carboxilato Reductasas/aislamiento & purificación , Spinacia oleracea/enzimología , Resinas Acrílicas , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Sulfato de Amonio/farmacología , Cloroplastos/enzimología , Etanolaminas , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Polímeros , Prolina/biosíntesis , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/metabolismo , Homología de Secuencia de Aminoácido , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and delta1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)(-1) and 4.3 nkat (mg protein)(-1), respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28.9 kDa as calculated from the sequence. Apparent Km values for PCA and NADH were 0.19 mM and 0.025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent Km values: 1.55 mM for proline and 10.5 mM for NAD at pH 10.0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.
Asunto(s)
Clostridium/enzimología , Prolina/biosíntesis , Pirrolina Carboxilato Reductasas/genética , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Secuencia de Aminoácidos , Clonación Molecular , Clostridium/genética , Clostridium/crecimiento & desarrollo , Genes Bacterianos , Datos de Secuencia Molecular , Peso Molecular , Ornitina/metabolismo , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
The activity of P5C metabolizing enzymes: OAT, P5CR, PO, and P5CD, in the camel tick Hyalomma dromedarii has been followed throughout embryogenesis. The profiles of enzymatic activity showed clear differences in the four enzymes as the embryos grew older. During purification of P5CD to homogeneity the ion exchange chromatography steps lead to two separate forms (termed A and B) with different molecular weights (60,000-59,000 and 50,000-52,000 for the native and denatured enzymes, respectively), amino acid composition, Km for P5C and coenzymes, varying dehydrogenase activities with different substrate specificity when supplied with various aldehyde substrates. Both P5CD A and B exhibited sharp optima at pH 7.5. The effect of different divalent cations and competitive and noncompetitive inhibitors was examined. The changes in P5C metabolizing enzymes during embryogenesis suggest that H. dromedarii has the metabolic potential to convert ornithine into proline and glutamate.
Asunto(s)
Pirroles/metabolismo , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Pirrolina Carboxilato Reductasas/metabolismo , Garrapatas/embriología , Garrapatas/enzimología , Nucleótidos de Adenina/farmacología , Aldehídos , Aminoácidos/análisis , Aminoácidos/farmacología , Animales , Cationes Bivalentes/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Peso Molecular , Óvulo/enzimología , Pirrolina Carboxilato Reductasas/química , Especificidad por Sustrato , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
Pyrrole-2-carboxylate oxygenase was purified 8.2-fold to homogeneity from Arthrobacter strain Py1 grown on pyrrole-2-carboxylate as sole carbon, nitrogen, and energy source. FAD and dithioerythritol had to be present during the purification procedure to stabilize the enzyme activity. The molecular mass of the pyrrole-2-carboxylate oxygenase was about 160 kDa by gel filtration chromatography and native gradient PAGE, only one polypeptide of about 60 kDa was present after SDS-PAGE. The FAD content was 2.7 to 3.6 mol FAD per enzyme (160 kDa). The non-covalently bound FAD of the pyrrole-2-carboxylate oxygenase was reduced by NADH and reoxidized by oxygen and pyrrole-2-carboxylate. The enzyme exhibited a narrow substrate specificity. Besides pyrrole-2-carboxylate, only pyrrole, pyrrole-2-aldehyde, and indole-2-carboxylate stimulated the oxygen consumption at a very low rate. The enzyme activity was strongly reduced by different sulfhydryl group inhibitors, but it could be restored by 2-mercaptoethanol or dithiothreitol. The content of pyrrole-2-carboxylate oxygenase was about 6% of the soluble protein as determined by antibodies raised against the enzyme. No cross reacting material was present in other bacteria also able to degrade pyrrole-2-carboxylate. A low amount of the enzyme was present in uninduced cells of Arthrobacter strain Py1, although the enzymatic activity was below the detection limit. The N-terminal amino acid sequence of the enzyme did not contain the consensus sequence GXGXXG found to be present close to the N-terminus of many flavin-dependent monoxygenases sequenced so far.
Asunto(s)
Arthrobacter/enzimología , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Secuencia de Aminoácidos , Western Blotting , Electroforesis en Gel de Poliacrilamida , Flavina-Adenina Dinucleótido/metabolismo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Consumo de Oxígeno/fisiología , Oxigenasas/metabolismo , Pirrolina Carboxilato Reductasas/análisis , Pirrolina Carboxilato Reductasas/antagonistas & inhibidores , Especificidad por SustratoRESUMEN
Electrophoretic evidence was obtained for two forms of pyrroline-5-carboxylate reductase (P5CR) in soybean nodules. One form was purified over 2300-fold. The apparent sizes of the polypeptides comprising the pyrroline-5-carboxylate reductases from soybean cytosol (29,700) and Escherichia coli (28,000) were consistent with those predicted from the sequences of the genes encoding them (Deutch et al., 1982 Nucleic Acid Res. 10, 7701-7714; Delauney and Verma, 1990 Mol. Gen. Genet. 221, 299-305). Primary structural analysis of the intact soybean P5CR subunit indicated that the amino-terminal residue is blocked. Analyses of a 12-mer and a 21-mer isolated from a cyanogen bromide digest were consistent with the proposition that the soybean P5CR isolated in these studies is very similar, although perhaps not identical, to the polypeptide predicted for the recently cloned soybean reductase (Delauney and Verma, 1990 Mol. Gen. Genet. 221, 299-305).
Asunto(s)
Glycine max/enzimología , Isoenzimas/aislamiento & purificación , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Secuencia de Aminoácidos , Cromatografía de Afinidad/métodos , Cromatografía DEAE-Celulosa/métodos , Electroforesis en Gel de Poliacrilamida , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/aislamiento & purificación , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/metabolismoRESUMEN
delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.
Asunto(s)
Cristalino/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Concentración de Iones de Hidrógeno , Cinética , Masculino , Peso Molecular , NAD/metabolismo , NADP/metabolismo , Pirrolina Carboxilato Reductasas/antagonistas & inhibidores , Pirrolina Carboxilato Reductasas/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The sequence of the Escherichia coli proC gene which encodes for delta 1-pyrroline-5-carboxylate (PCA) reductase was determined. Overproduction of the proC gene product via an expression plasmid carrying the bacteriophage lambda PL promoter allowed the purification to homogeneity of PCA reductase by affinity adsorption chromatography. NH2 and COOH-terminal analysis and amino acid composition of the purified proC protein is consistent with the gene sequence reported. The molecular weight of the proC monomer is 28,112.
Asunto(s)
Escherichia coli/enzimología , Genes , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Pirrolina Carboxilato Reductasas/genética , Secuencia de Aminoácidos , Bacteriófago lambda/genética , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Recombinante/metabolismo , Escherichia coli/genética , Peso Molecular , Plásmidos , Pirrolina Carboxilato Reductasas/aislamiento & purificaciónRESUMEN
In this paper we report the purification of a protein which is able to catalyze both the proline oxidase and the pyrroline-5-carboxylic acid dehydrogenase activities necessary for the oxidation of proline to glutamic acid. The purification involves the preparation of a crude membrane pellet, detergent solubilization, ammonium sulfate fractionation, and DEAE-chromatography. We are able to obtain an essentially pure preparation (greater than 95% pure) after only a 52-fold purification, demonstrating that the protein is a major protein in cells fully induced for proline utilization. Both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-purity throughout our purification. Velocity sedimentation of the purified protein demonstrates that both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-sediment. Early in the purification procedure we are able to detect two species of protein which have both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities. Our procedure purifies only the larger molecular weight species. The purified protein is a dimer composed of identical 132,000-dalton subunits. Analysis of mutants defective for proline utilization demonstrate that the bifunctional enzyme is the putA gene product.
Asunto(s)
Proteínas Bacterianas/genética , Genes , Proteínas de la Membrana/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Prolina Oxidasa/genética , Pirrolina Carboxilato Reductasas/genética , Salmonella typhimurium/enzimología , 1-Pirrolina-5-Carboxilato Deshidrogenasa , Proteínas Bacterianas/aislamiento & purificación , Membrana Celular/enzimología , Proteínas de la Membrana/aislamiento & purificación , Peso Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Prolina Oxidasa/aislamiento & purificación , Pirrolina Carboxilato Reductasas/aislamiento & purificaciónRESUMEN
A homogeneous preparation of delta 1-pyrroline-5-carboxylate reductase (L-proline: NAD (P)+ 5-oxidoreductase, EC 1.5.1.2) was obtained from baker's yeast by an affinity chromatography, using 5' AMP-Sepharose 4B. After the 1st DEAE-Sephadex column chromatography, the enzyme absorbed on 5' AMP-Sepharose column was eluted with 1 mM ATP. The final preparation was homogeneous on polyacrylamide gel electrophoresis and purified 3500-fold from the crude extract. This purified enzyme was very useful as a coupling enzyme for the assays of ornithine and pyrroline-5-carboxylate in tissues, and for the rate assay of ornithine aminotransferase activity.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Adenosina Monofosfato , Cromatografía de Afinidad , SefarosaRESUMEN
delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) from Baker's yeast has been purified and characterized. Purification to an apparently homogenous protein was effected by using 'reagent-grade' water containing dithiothreitol and by maintaining a constant pH 7.5, because of the instability of the enzyme protein. The enzyme was purified approximately 200-fold from the crude extract of baker's yeast, and it is a negatively-charged protein with a molecular weight of 125 000, containing an active SH group which participates in binding with NAD(P)H. The Km value for DL-pyrroline-5-carboxylate is 0.8 x 10(-4) M; for NADH, it is 4.8 x 10(-5) M; and for NADPH, it is 5.6 x 10(-5) M. These Km values are much smaller than those of enzymes from other sources. The purified enzyme is free of contaminating enzymes which might interfere with its use in assays. The enzyme has been applied successfully to the assays of L-delta 1-pyrroline-5-carboxylate and L-ornithine in tissue, and in vivo levels of these amino acids in rat liver are reported.
Asunto(s)
Ornitina/análisis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Pirroles/análisis , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Animales , Hígado/análisis , RatasRESUMEN
gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Oxidorreductasas/metabolismo , Pirrolina Carboxilato Reductasas/metabolismo , Genes , Cinética , Mutación , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/aislamiento & purificación , Prolina/biosíntesis , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Pirrolina Carboxilato Reductasas/antagonistas & inhibidores , Pirrolina Carboxilato Reductasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
Proline dehydrogenase and delta1-pyrroline-5-carboxylic acid (PCA) reductase activities were copurified 60- and 130-fold, respectively, from extracts of Clostridium sporogenes. The primary change in the ratio of activites was the result of a loss of proline dehydrogenase activity during dialysis. Both activities were eluted in single peaks from diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200 columns. They had identical sedimentation coefficients (10.3S), as determined in linear sucrose gradients, and identical isoelectric points (4.95 to 5.12) based on isoelectric focusing. The proline dehydrogenase activity was dependent on nicotinamide adenine dinucleotide and L-proline, and the PCA reductase required L-PCA and reduced nicotinamide adenine dinucleotide. The optimum pH for the assay of proline dehydrogenase was approximately 10.2, whereas that for PCA reductase was 6.5 to 7.5. An increase in pH from 8.0 to 10.2 greatly decreased the apparent Michaelis constant observed for L-proline, and an increase from pH 8.3 to 8.6 resulted in a large shift in the reaction equilibrium toward PCA. Both the dehydrogenase and reductase activities were stabilized to heating at 65 degrees C for 5 min by solutes of high ionic strength and were inactivated in a similar fashion when dissolved in low-ionic-strength buffer. The specific activities for both were reduced by about 50% when glucose was added to the growth medium. The data support the conclusion that L-proline and L-PCA are interconverted by either a single enzyme or an enzyme complex in extracts of C. sporogenes cells.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Clostridium/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Pirrolina Carboxilato Reductasas/metabolismo , Aminoácido Oxidorreductasas/aislamiento & purificación , Centrifugación por Gradiente de Densidad , Glucosa/farmacología , Calor , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Prolina , Pirrolina Carboxilato Reductasas/aislamiento & purificaciónRESUMEN
delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.