Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.49 (phosphoenolpyruvate carboxykinase)
4,654 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.
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PMID:Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium. 309 61

Determination of whether CO2 or HCO3- is the substrate for an enzymatic carboxylation has generally been accomplished by taking advantage of the fact that equilibration of these two compounds requires more than a minute at temperatures below 15 degrees C; thus different kinetics of carboxylation are obtained depending on whether CO2 or HCO3- is used to initiate the reaction. We report a new method using 13C18O2 as substrate for determining the CO2/HCO3- specificity of carboxylases. If CO2 is the substrate, then the 18O content of the 13C-containing product is the same as that of the 13CO2 used, whereas if HCO3- is the substrate, the 18O content is 2/3 that of the starting material. The method is independent of the detailed kinetics of the CO2/HCO3- interconversion and independent of the presence of contaminating unlabeled CO2 or HCO3-. Isotopic analysis is accomplished by 13C NMR. The method has been used to confirm that HCO3- is the substrate for phosphoenolpyruvate carboxylase. Studies of oxygen-18 isotope shifts in phosphorus NMR spectra have permitted confirmation of the observation that label is transferred from HC18O3- into Pi during the carboxylation of phosphoenolpyruvate.
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PMID:Determination of substrate specificity of carboxylases by nuclear magnetic resonance. 311 Dec 98

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas. 312 Jul 92

A rapid, continuous spectrophotometric method has been developed for the assay of decarboxylases. The assay uses a coupled enzyme system in which liberated CO2 is reacted with phosphoenolpyruvate and phosphoenolpyruvate carboxylase to form oxaloacetate, which in turn is reduced by malate dehydrogenase to L-malate concomitantly with the oxidation of NADH to NAD. The resultant decrease in absorbance at 340 nm accurately reflects the activity of the decarboxylase. The method is capable of detecting the liberation of as little as 1 nmol of CO2/min and was tested in assays of lysine decarboxylase, orotidine-5'-phosphate decarboxylase, and 4'-phosphopantothenoyl-L-cysteine decarboxylase.
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PMID:A general coupled spectrophotometric assay for decarboxylases. 313 80

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.
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PMID:Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos. 313 24

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.
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PMID:Pathway and sites for energy conservation in the metabolism of glucose by Selenomonas ruminantium. 314 85

Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.
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PMID:Presence of anaplerotic reactions and transamination, and the absence of the tricarboxylic acid cycle in mollicutes. 314 76

The conversions of the isotope from [1-14C]acetate, [1-14C]glucose and [6-14C]glucose to CO2 and fatty acids in acini isolated from the mammary gland at the peak of lactation were studied. The incorporation of [9,10-3H]oleate into triacylglycerol synthesis as single substrate or in combination with substrates that potentially may supply trioses-phosphate was also determined. The rate of fatty acid synthesis paralleled the activity of the hexose monophosphate shunt and the data obtained reveal that little carbon from triose stage enters the phosphohexose pool via reversal of glycolytic pathway. The results are interpreted in terms of the NADPH producing systems and phosphoenolpyruvate carboxykinase activities as well as the possible implications in lipogenic and glyceroneogenic pathways.
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PMID:Glycerogenic pathway in the rat mammary gland. 356 49

The isozymic forms of maize phosphoenolpyruvate carboxylase (P-enolpyruvate carboxylase) involved in photosynthetic CO2 fixation were shown by protein gel blot analysis to consist of 100-kDa subunits. The nonautotrophic isoform found in roots is comprised of 96-kDa subunits and is about 50-100-fold less prevalent. Further analysis of P-enolpyruvate carboxylase isoforms made use of cloned cDNA probes. Two cDNA clones were isolated from a library constructed from maize leaf poly(A) RNA. The largest clone was complementary to about 25% of P-enolpyruvate carboxylase mRNA, which is 3.4 kilobases in length. The quantity of P-enolpyruvate carboxylase mRNA in green, mature leaf tissue was estimated to be 0.20% of poly(A) RNA, whereas P-enolpyruvate carboxylase mRNA in roots was about 100-fold less prevalent. We used thermal denaturation of a P-enolpyruvate carboxylase cDNA probe hybridized to RNA gel blots to estimate the degree of sequence difference between mRNAs encoding different P-enolpyruvate carboxylase isoforms. There appear to be at least two prevalent P-enolpyruvate carboxylase mRNAs in green leaves which are significantly different in sequence, as are P-enolpyruvate carboxylase mRNAs in roots and shoots. The hybridization pattern of maize genomic DNA Southern blots indicates that P-enolpyruvate carboxylase is encoded by a small gene family.
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PMID:Maize phosphoenolpyruvate carboxylase. Cloning and characterization of mRNAs encoding isozymic forms. 370 Mar 88

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.
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PMID:Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver. 378 66


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