Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
 4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The metabolism of L-[U-14C]aspartate, L-[1-14C]aspartate and L-[4-14C]aspartate was studied in isolated guinea-pig kidney tubules. 2. Oxidation of C-1 plus that of C-4 of aspartate accounted for 90-92% of the CO2 released from aspartate, whereas oxidation of the inner carbon atoms of aspartate (which occurs beyond the 2-oxoglutarate dehydrogenase step) represented only 8-10% of aspartate carbon oxidation. 3. The formation of [1-14C]glutamine and [1-14C]glutamate from [1-14C]aspartate and [4-14C]aspartate indicated that about one-third of the oxaloacetate synthesized from aspartate underwent randomization at the level of fumarate. 4. With [U-14C]aspartate as substrate, the percentage of the C-1 of glutamate and glutamine found radiolabelled after 60 min of incubation was 92.7% and 47.5% in the absence and the presence of bicarbonate respectively. 5. That CO2 fixation occurred at high rates in the presence of bicarbonate was demonstrated by incubating tubules with aspartate plus [14C]bicarbonate; under this condition, the label fixed was found in C-1 of glutamate, glutamine and aspartate, as well as in C-4 of aspartate, demonstrating not only randomization of aspartate carbon but also aspartate resynthesis secondary to oxaloacetate cycling via phosphoenolpyruvate carboxykinase, pyruvate kinase and pyruvate carboxylase. 6. The importance of CO2 fixation in glutamine synthesis from aspartate is discussed in relation to the possible role of the guinea-pig kidney in systemic acid-base regulation in vivo.
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PMID:Release and fixation of CO2 by guinea-pig kidney tubules metabolizing aspartate. 132 Mar 75

The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.
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PMID:Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1. 172 25

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

Evidence is presented which suggests that Methylobacterium organophilum contains isoenzymes of phosphoenolpyruvate carboxylase activity. Methanol-grown cells contained an acetyl coenzyme A (CoA)-insensitive activity which precipitated in a 65 to 75% of saturation ammonium sulfate fraction. Succinate-grown cells contained an acetyl-CoA-stimulated activity which precipitated in a 55 to 65% of saturation ammonium sulfate fraction. Mutants unable to grow on methanol appeared to lack acetyl-CoA-insensitive activity. This acetyl-CoA-insensitive phosphoenolpyruvate carboxylase, along with malyl-CoA lyase, is proposed to be encoded by the C-1 operon. The gene for formate dehydrogenase appeared to reside outside the operon and was not inducible by methanol. M. organophilum was unable to grow on formate, and evidence is presented suggesting that formate is unable to induce the enzymes which comprise the serine pathway for formaldehyde fixation. An expanded model for the C-1 operon is presented.
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PMID:Extension of the model concerning linkage of genes coding for C-1 related functions in Methylobacterium organophilum. 678 18

Previous studies indicated that in pancreatic islets the amount of glucose-derived pyruvate that enters mitochondrial metabolism via carboxylation is approximately equal to that entering via decarboxylation and that both carboxylation and decarboxylation are correlated with capacitation of glucose metabolism and insulin release. The relatively high rate of carboxylation is consistent with the current study's finding that pyruvate carboxylase is as abundant in pancreatic islets as it is in liver and kidney. Since islets do not contain phosphoenolpyruvate carboxykinase and, therefore, cannot carry out glyconeogenesis from pyruvate, the carboxylase might be present in the islet to participate in novel anaplerotic reactions. This idea was first explored by incubating mitochondria from various tissues with pyruvate. Mitochondria from tissues, such as pancreatic islets, liver, and kidney, in which pyruvate carboxylase is abundant, exported a large amount of malate and little or no citrate, isocitrate, and aspartate to the medium. The amount of malate within the mitochondria was < 1% that in the medium. When pancreatic islet mitochondria were incubated with [1-14C]pyruvate, radioactive carbon appeared in the medium primarily in malate. Very little radioactivity appeared in amino acids, and little or no radioactivity appeared in citrate and isocitrate. Carbon 1 of pyruvate can be incorporated into malate and other citric acid cycle intermediates only via carboxylation, as this carbon would be lost via decarboxylation when pyruvate enters the citric acid cycle as acetyl-CoA via the pyruvate dehydrogenase reaction. The amount of malate formed equaled the 14CO2 formed and the radioactivity from C-1 of pyruvate recovered in malate slightly exceeded the formation of 14CO2 in agreement with our previous studies that reported a high rate of carboxylation of pyruvate in intact islets. When intact pancreatic islets were incubated with methyl [U-14C]succinate as a mitochondrial source of four-carbon dicarboxylic acids, radioactivity appeared in pyruvate and lactate. Taken together with previous studies, the current results suggest that during glucose-induced insulin secretion there is a shuttle operating across the mitochondrial membrane in which glucose-derived pyruvate is taken up by mitochondria and carboxylated to oxaloacetate by pyruvate carboxylase. The oxaloacetate is converted to malate which exits the mitochondrion, where, in the cytosol, it is decarboxylated to pyruvate in the reaction catalyzed by malic enzyme. This pyruvate re-enters mitochondrial pools. Such a cycle produces NADPH in the cytosol. Since it is a cycle, this shuttle can produce far more NADPH than the pentose phosphate pathway, which is known to be a very minor route of glucose metabolism in the islet.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets. Further implication of cytosolic NADPH in insulin secretion. 765 22

We report a new continuous spectrophotometric assay for human cystathionine beta-synthase (hCBS). This assay relies upon the finding that hCBS will take cysteamine in place of L-homocysteine, thereby producing thialysine. Thialysine is, in turn, decarboxylated by lysine decarboxylase, releasing CO2 that is monitored by the sequential action of phosphoenolpyruvate carboxylase and L-malate dehydrogenase. The decrease in absorbance at 340 nm is monitored as reduced nicotinamide adenine dinucleotide is consumed. Using this four-enzyme couple, we find that Km(app) = 1.2+/-0.2 mM for L-serine and 5.6+/-2.2 mM for cysteamine, with kcat = 1.3+/-0.1s(-1) for the formation of thialysine by hCBS. For comparison purposes, the same hCBS reaction was monitored via a radioactive single time point assay using 14C-(C-1)-labeled L-serine and cysteamine as substrates, counting the thialysine product, following ion exchange chromatography. This assay yielded Km(app) = 2.2+/-0.5 mM for L-serine and 6.6+/-2.2 for cysteamine, with kcat = 2.5+/-0.4 s(-1). These numbers indicate that, although it possesses a shortened carbon chain and lacks a carboxyl group, cysteamine displays a catalytic efficiency (kcat/Km) with hCBS that is within an order of magnitude of that observed with its natural thiol cosubstrate, L-homocysteine.
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PMID:A continuous spectrophotometric assay for human cystathionine beta-synthase. 1595 86

Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo (13)C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO(2) fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO(2) acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the alpha-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.
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PMID:Insights into the autotrophic CO2 fixation pathway of the archaeon Ignicoccus hospitalis: comprehensive analysis of the central carbon metabolism. 1740 Jul 48