Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.4.1.1 (pyruvate carboxylase)
1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of temperature on the relationships among the rates of pyruvate carboxylation, O(2) uptake (J(o)), oxidative phosphorylation (J(p)), and the free energy of ATP hydrolysis (G(p)) were studied in liver mitochondria isolated from 250-g female rats. Pyruvate carboxylation was evaluated at 37, 40, and 43 degrees C. In disrupted mitochondria, pyruvate carboxylase maximal reaction velocity increased from 37 to 43 degrees C with an apparent Q(10) of 2.25. A reduction in ATP/ADP ratio decreased enzyme activity at all three temperatures. In contrast, in intact mitochondria, increasing temperature failed to increase pyruvate carboxylation (malate + citrate accumulation) but did result in increased J(o) and decreased extramitochondrial G(p). J(p) was studied in respiring mitochondria at 37 and 43 degrees C at various fractions of state 3 respiration, elicited with a glucose + hexokinase ADP-regenerating system. The relationship between J(o) and G(p) was similar at both temperatures. However, hyperthermia (43 degrees C) reduced the J(p)/J(o) ratio, resulting in lower G(p) for a given J(p). Fluorescent measurements of membrane phospholipid polarization revealed a transition in membrane order between 40 and 43 degrees C, a finding consistent with increased membrane proton conductance. It is concluded that hyperthermia augments nonspecific proton leaking across the inner mitochondrial membrane, and the resultant degraded energy state offsets temperature stimulation of pyruvate carboxylase. As a consequence, at high temperatures approaching 43 degrees C, the pyruvate carboxylation rate of intact liver mitochondria may fail to exhibit a Q(10) effect.
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PMID:Hyperthermia impairs liver mitochondrial function in vitro. 1080 Dec 93

This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium. The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg(2+), Co(2+), and Mn(2+), by aspartate, but not by glutamate and alpha-ketoglutarate. Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observations would not fit the idea that the M. smegmatis enzyme fulfills a straightforward anaplerotic function; in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme. Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol. The PYCs of M. smegmatis and Mycobacterium tuberculosis were highly homologous to each other. In M. smegmatis, M. tuberculosis and M. lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in an identical operon-like arrangement. Thus, M. smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria. Also, the ease of purification and the extraordinary stability could make the M. smegmatis enzyme a model for studying the structure-function relationships of PYCs in general. It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya.
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PMID:Pyruvate carboxylase from Mycobacterium smegmatis: stabilization, rapid purification, molecular and biochemical characterization and regulation of the cellular level. 1091 17

The pyruvate carboxylase (PYC) of the hyperthermophilic, strictly hydrogenotrophic, autotrophic and marine methanarchaeon Methanococcus jannaschii was purified to homogeneity. Optimal activity was at pH 8.5, > or = 80 degrees C, and a KCl concentration of 0.175 M. This enzyme is the most thermophilic PYC so far studied. Unlike the Methanobacterium thermoautotrophicum enzyme, Mc. jannaschii PYC was expressed in cells grown without an external source of biotin and in the purified form was stable during storage at 4, -20 and -80 degrees C. However, it was rapidly inactivated at 80 degrees C. The enzyme was insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and was strongly inhibited by ATP and ADP (apparent Km, for ATP, 0.374 +/- 0.039 mM; apparent Ki for ATP, 5.34 +/- 2.14 mM; Ki for ADP, 0.89 +/- 0.18 mM). It was also strongly inhibited when the Mg2+ concentration in the assay exceeded that of ATP. Thus, this stable PYC could serve as a model for mechanistic studies on archaeal PYCs. It was apparently an alpha4beta4-type PYC composed of a non-biotinylated 55.5-kDa subunit (PYCA) and a 64.2-kDa biotinylated subunit (PYCB). The determined NH2-terminal sequences for these subunits provided additional support for our earlier proposal to rename the ORFs MJ1229 and MJ1231 in the NCBI Mc. jannaschii genome sequence database as PYCA and PYCB, respectively; even very recently, these have been misidentified as a subunit of acetyl-CoA carbxoylase (AccC) and the alpha-subunit of ion-pumping oxaloacetate decarboxylase (OADalpha), respectively.
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PMID:A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii. 1119 96

This is the first report on a bacterial verterbrate-type GTP-dependent phosphoenolpyruvate carboxykinase (PCK). The pck gene of Mycobacterium smegmatis was cloned. The recombinant PCK was overexpressed in Escherichia coli in a soluble form and with high activity. The purified enzyme was found to be monomeric (72 kDa), thermophilic (optimum temperature, 70 degrees C), very stable upon storage at 4 degrees C, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by alpha-ketoglutarate. The requirement for a divalent cation for activity was fulfilled best by Mn(2+) and Co(2+) and poorly by Mg(2+). At 37 degrees C, the highest V(m) value (32.5 units/mg) was recorded with Mn(2+) and in the presence of 37 mm dithiothreitol (DTT). The presence of Mg(2+) (2 mm) greatly lowered the apparent K(m) values for Mn(2+) (by 144-fold in the presence of DTT and by 9.4-fold in the absence of DTT) and Co(2+) (by 230-fold). In the absence of DTT but in the presence of Mg(2+) (2 mm) as the co-divalent cation, Co(2+) was 21-fold more efficient than Mn(2+). For producing oxaloacetate, the enzyme utilized both GDP and IDP; ADP served very poorly. The apparent K(m) values for phosphoenolpyruvate, GDP, and bicarbonate were >100, 66, and 8300 micrometer, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 12 microm, respectively. Thus, this enzyme preferred the gluconeogenesis/glycerogenesis direction. This property fits the suggestion that in M. smegmatis, pyruvate carboxylase is not anaplerotic but rather gluconeogenic (Mukhopadhyay, B., and Purwantini, E. (2000) Biochim. Biophys. Acta. 1475, 191-206). Both in primary structure and kinetic properties, the mycobacterial PCK was very similar to its vertebrate-liver counterparts and thus could serve as a model for these enzymes; examples for several immediate targets are presented.
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PMID:A GTP-dependent vertebrate-type phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis. 1127 51

Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO(2) fixation reaction is catalyzed by an apparent alpha(4)beta(4)-type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits. The purified enzyme was most active at 70 degrees C, insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg(2+). It showed negative cooperativity towards bicarbonate at 70 degrees C but not at 37 degrees C. The organism expressed holo-PYC without an external supply of biotin and, thus, synthesized biotin. pycA, pycB, and a putative bpl gene formed a novel operon-like arrangement. Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of M. barkeri and the closely related euryarchaeon Archaeoglobus fulgidus appeared to be of the Escherichia coli-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL). We found the element Tyr(Phe)ProX(5)Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their "swinging arms."
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PMID:Oxaloacetate synthesis in the methanarchaeon Methanosarcina barkeri: pyruvate carboxylase genes and a putative Escherichia coli-type bifunctional biotin protein ligase gene (bpl/birA) exhibit a unique organization. 1137 47

The biochemical events associated with the onset of lipid accumulation in Mucor circinelloides and Mortierella alpina, under conditions of nitrogen-limited growth, have been elucidated; they differ in key aspects from those described in oleaginous yeasts. The NAD+:isocitrate dehydrogenases of Mc. circinelloides and Mort. alpina were not absolutely dependent on AMP for activity. Furthermore, changes in the cellular adenine nucleotide pools and energy charge were different from those reported for oleaginous yeasts. In Mc. circinelloides ATP, ADP and AMP concentrations all decreased by 50% after nitrogen limitation, leading to a constant energy charge at the expense of the size of the total adenylate pool. Pyruvate carboxylase in Mc. circinelloides was cytosolic, having implications for the organization of lipid synthesis in filamentous fungi. As a result of the data obtained, a revised and more concerted mechanism for the initiation of storage lipid accumulation is put forward for filamentous fungi.
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PMID:Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. 1157 64

The effects of benfluorex and two of its metabolites (S 422-1 and S 1475-1) on fatty acid and glucose metabolic fluxes and specific gene expression were studied in hepatocytes isolated from 24-h fasted rats. Both benfluorex and S 422-1 (0.1 or 1 mmol/l) reduced beta-oxidation rates and ketogenesis, whereas S 1475-1 had no effect. At the same concentration, benfluorex and S 422-1 were more efficient in reducing gluconeogenesis from lactate/pyruvate than S 1475-1. Benfluorex inhibited gluconeogenesis at the level of pyruvate carboxylase (45% fall in acetyl-CoA concentration) and of glyceraldehyde-3-phosphate dehydrogenase (decrease in ATP/ADP and NAD(+)/NADH ratios). Accordingly, neither benfluorex nor S 422-1 inhibited gluconeogenesis from dihydroxyacetone, but both stimulated gluconeogenesis from glycerol. In hepatocytes cultured in the presence of benfluorex or S 422-1 (10 or 100 micromol/l), the expression of genes encoding enzymes of fatty acid oxidation (carnitine palmitoyltransferase [CPT] I), ketogenesis (hydroxymethylglutaryl-CoA synthase), and gluconeogenesis (glucose-6-phosphatase, PEPCK) was decreased, whereas mRNAs encoding glucokinase and pyruvate kinase were increased. By contrast, Glut-2, acyl-CoA synthetase, and CPT II gene expression was not affected by benfluorex or S 422-1. In conclusion, this work suggests that benfluorex mainly via S 422-1 reduces gluconeogenesis by affecting gene expression and metabolic status of hepatocytes.
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PMID:Effects of benfluorex on fatty acid and glucose metabolism in isolated rat hepatocytes: from metabolic fluxes to gene expression. 1214 46

The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.
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PMID:Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A. 1474 53

Because in the phloem sap of maize (Zea mays L.) leaves a quarter of the total amino nitrogen can be found as alanine, the capacity of a de novo synthesis of alanine from 3-phosphoglycerate (3-PGA) was studied with isolated bundle sheath (BS) strands of maize. Inasmuch as these cells have retained their plasmodesmatic openings, it was possible to study the formation of alanine from 3-PGA when glutamate and ADP were being added. Alanine synthesis required the existence of the intact cell structure. From the formation of the intermediates, partially released to the medium, the activities of the enzymes of the reaction chain from 3-PGA to alanine could be measured in the intact cells. The results show that in the BS cells the rate of alanine production from pyruvate (0.5 micromole/minute per milligram BS chlorophyll) is more than sufficient to produce one-fourth of the assimilated nitrogen as alanine. As the activity of pyruvate kinase in intact bundle sheath cells in the light was found to be only 0.2 micromole/minute per milligram BS chlorophyll, it is concluded that in the light part of the conversion of 3-PGA to pyruvate may not occur via pyruvate kinase reaction, but via phosphoeno/pyruvate carboxylase, NADP-malate dehydrogenase, and NADP-malic enzyme in the mesophyll and BS cells.
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PMID:Alanine synthesis by bundle sheath cells of maize. 1666 62

A mathematical model of the perfused rat liver was developed to predict intermediate metabolite concentrations and fluxes in response to changes in various substrate concentrations in the perfusion medium. The model simulates gluconeogenesis in the liver perfused separately with lactate and pyruvate and the combination of these substrates with fatty acids (oleate). The model consists of key reactions representing gluconeogenesis, glycolysis, fatty acid metabolism, tricarboxylic acid cycle, oxidative phosphorylation, and ketogenesis. Michaelis-Menten-type kinetic expressions, with control by ATP/ADP, are used for many of the reactions. For key regulated reactions (fructose-1,6-bisphosphatase, phosphofructokinase, pyruvate carboxylase, pyruvate dehydrogenase complex, and pyruvate kinase), rate expressions were developed that incorporate allosteric effectors, specific substrate relationships (e.g., cooperative binding), and/or phosphorylation/dephosphorylation using in vitro enzyme activity data and knowledge of the specific mechanisms. The model was independently validated by comparing model predictions with 10 sets of experimental data from 7 different published works, with no parameter adjustments. The simulations predict the same trends, in terms of stimulation of substrate uptake by fatty acid addition, as observed experimentally. In general, the major metabolic indicators calculated by the model are in good agreement with experimental results. For example, the simulated glucose/pyruvate mass yield is 43% compared with the average of 45% reported in the literature. The model accurately predicts the specific time constants of the glucose response (2.5-4 min) and the dynamic behavior of substrate and product fluxes. It is expected that this model will be a useful tool for analyzing the complex relationships between carbohydrate and fat metabolism.
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PMID:A computer model of gluconeogenesis and lipid metabolism in the perfused liver. 1791 49


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