Showing papers on "Pyruvate kinase published in 2002"

Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

Abstract: The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

Pyruvate kinase type M2: a crossroad in the tumor metabolome

Abstract: Cell proliferation is a process that consumes large amounts of energy. A reduction in the nutrient supply can lead to cell death by ATP depletion, if cell proliferation is not limited. A key sensor for this regulation is the glycolytic enzyme pyruvate kinase, which determines whether glucose carbons are channelled to synthetic processes or used for glycolytic energy production. In unicellular organisms pyruvate kinase is regulated by ATP, ADP and AMP, by ribose 5-P, the precursor of the nucleic acid synthesis, and by the glycolytic intermediate fructose 1,6-P2 (FBP), thereby adapting cell proliferation to nutrient supply. The mammalian pyruvate kinase isoenzyme type M2 (M2-PK) displays the same kinetic properties as the pyruvate kinase enzyme from unicellular organisms. The mammalian M2-PK isoenzyme can switch between a less active dimeric form and a highly active tetrameric form which regulates the channeling of glucose carbons either to synthetic processes (dimeric form) or to glycolytic energy production (tetrameric form). Tumor cells are usually characterized by a high amount of the dimeric form leading to a strong accumulation of all glycolytic phosphometabolites above pyruvate kinase. The tetramer-dimer ratio is regulated by ATP, FBP and serine and by direct interactions with different oncoproteins (pp60v-src, HPV-16 E7). In solid tumors with sufficient oxygen supply pyruvate is supplied by glutaminolysis. Pyruvate produced in glycolysis and glutaminolysis is used for the synthesis of lactate, glutamate and fatty acids thereby releasing the hydrogen produced in the glycolytic glyceraldehyde 3-phosphate dehydrogenase reaction.

Analysis of gluconeogenic and anaplerotic enzymes in Campylobacter jejuni: an essential role for phosphoenolpyruvate carboxykinase

Abstract: Campylobacter jejuni is unable to utilize glucose as a carbon source due to the absence of the key glycolytic enzyme 6-phosphofructokinase. The genome sequence of C. jejuni NCTC 11168 indicates that homologues of all the appropriate enzymes for gluconeogenesis from phosphoenolpyruvate (PEP) are present, in addition to the anaplerotic enzymes pyruvate carboxylase (PYC), phosphoenolpyruvate carboxykinase (PCK) and malic enzyme (MEZ). Surprisingly, a pyruvate kinase (PYK) homologue is also present. To ascertain the role of these enzymes, insertion mutants in pycA, pycB, pyk and mez were generated. However, this could not be achieved for pckA, indicating that PCK is an essential enzyme in C. jejuni. The lack of PEP synthase and pyruvate orthophosphate dikinase activities confirmed a unique role for PCK in PEP synthesis. The pycA mutant was unable to grow in defined medium with pyruvate or lactate as the major carbon source, thus indicating an important role for PYC in anaplerosis. Sequence and biochemical data indicate that the PYC of C. jejuni is a member of the alpha4beta4, acetyl-CoA-independent class of PYCs, with a 65.8 kDa subunit containing the biotin moiety. Whereas growth of the mez mutant was comparable to that of the wild-type, the pyk mutant displayed a decreased growth rate in complex medium. Nevertheless, the mez and pyk mutants were able to grow with pyruvate, lactate or malate as carbon sources in defined medium. PYK was present in cell extracts at a much higher specific activity [>800 nmol x min(-1) x (mg protein)(-1)] than PYC or PCK [<65 nmol x min(-1) x (mg protein)(-1)], was activated by fructose 1,6-bisphosphate and displayed other regulatory properties strongly indicative of a catabolic role. It is concluded that PYK may function in the catabolism of unidentified substrates which are metabolized through PEP. In view of the high K(m) of MEZ for malate (approximately 9 mM) and the lack of a growth phenotype of the mez mutant, MEZ seems to have only a minor anaplerotic role in C. jejuni.

Global Gene Expression Differences Associated with Changes in Glycolytic Flux and Growth Rate in Escherichia coli during the Fermentation of Glucose and Xylose

Abstract: The simplicity of the fermentation process (anaerobic with pH, temperature, and agitation control) in ethanologenic Escherichia coli KO11 and LY01 makes this an attractive system to investigate the utility of gene arrays for biotechnology applications. By using this system, gene expression, glycolytic flux, and growth rate have been compared in glucose-grown and xylose-grown cells. Although the initial metabolic steps differ, ethanol yields from both sugars were essentially identical on a weight basis, and little carbon was diverted to biosynthesis. Expression of only 27 genes changed by more than 2-fold in both strains. These included induction of xylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XylR and the cyclic AMP-CRP system and repression of Mlc-regulated genes encoding glucose uptake (ptsHIcrr, ptsG) and mannose uptake (manXYZ) during growth on xylose. However, expression of genes encoding central carbon metabolism and biosynthesis differed by less than 2-fold. Simple statistical methods were used to investigate these more subtle changes. The reproducibility (coefficient of variation of 12%) of expression measurements (mRNA as cDNA) was found to be similar to that typically observed for in vitro measurements of enzyme activities. Using Student's t test, many smaller but significant sugar-dependent changes were identified (p < 0.05 in both strains). A total of 276 genes were more highly expressed during growth on xylose; 307 genes were more highly expressed with glucose. Slower growth (lower ATP yield) on xylose was accompanied by decreased expression of 62 genes concerned with the biosynthesis of small molecules (amino acids, nucleotides, cofactors, and lipids), transcription, and translation; 5 such genes were expressed at a higher level. In xylose-grown cells, 90 genes associated with the transport, catabolism, and regulation of pathways for alternative carbon sources were expressed at higher levels than in glucose-grown cells, consistent with a relaxation of control by the cyclic AMP-CRP regulatory system. Changes in expression of genes encoding the Embden-Meyerhof-Parnas (EMP) pathway were in excellent agreement with calculated changes in flux for individual metabolites. Flux through all but one step, pyruvate kinase, was predicted to be higher during glucose fermentation. Expression levels (glucose/xylose) were higher in glucose-grown cells for all EMP genes except the isoenzymes encoding pyruvate kinase (pykA and pykF). Expression of both isoenzymes was generally higher during xylose fermentation but statistically higher in both strains only for pykF encoding the isoenzyme activated by fructose-6-phosphate, a key metabolite connecting pentose metabolism to the EMP pathway. The coordinated changes in expression of genes encoding the EMP pathway suggest the presence of a common regulatory system and that flux control within the EMP pathway may be broadly distributed. In contrast, expression levels for genes encoding the Pentose-Phosphate pathway did not differ significantly between glucose-grown and xylose-grown cells.

Genotoxic exposure is associated with alterations in glucose uptake and metabolism.

Abstract: Recent observations suggest that growth factor withdrawal can promote cell death in part through modulation of basic cellular bioenergetic pathways, including inhibition of glucose uptake and glycolytic metabolism. Whether DNA damage-initiated cell death pathways also involve bioenergetic deregulation has not been studied previously. Subtractive suppressive hybridization was used to identify changes in gene expression in murine cells after exposure to genotoxic stimuli, including cisplatin, etoposide, and γ-radiation. Among the genes identified in this screen were several that regulate glycolytic metabolism. Enzymes that catalyze key regulatory steps of glycolysis, including hexokinase, phosphofructokinase, and pyruvate kinase, appeared to be coordinately down-regulated by genotoxic exposure. Northern blotting confirmed that these changes in gene expression occur within 4 h of exposure to several DNA-damaging agents. Genotoxic exposure was found to similarly inhibit expression of both glut-1 and glut-3, genes that encode critical regulators of glucose uptake. Direct measurement of glycolytic rate and of oxygen consumption confirmed that genotoxic exposure resulted in suppression of both anaerobic and aerobic metabolism. Many of these metabolic changes mimic those observed after growth factor withdrawal. Together, these observations suggest that multiple apoptotic triggers, including growth factor withdrawal and genotoxic exposure, suppress cellular bioenergetic pathways. Mitochondrial responses to the resulting rapid decrease in metabolic substrates may play an important role in initiation of apoptotic cell death.

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

Abstract: The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit ∼0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate.

Experimental determination of control of glycolysis in Lactococcus lactis

Abstract: The understanding of control of metabolic processes requires quantitative studies of the importance of the different enzymatic steps for the magnitude of metabolic fluxes and metabolite concentrations. An important element in such studies is the modulation of enzyme activities in small steps above and below the wild-type level. We review a genetic approach that is well suited for both Metabolic Optimization and Metabolic Control Analysis and studies on the importance of a number of glycolytic enzymes for metabolic fluxes in Lactococcus lactis. The glycolytic enzymes phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and lactate dehydrogenase (LDH) are shown to have no significant control on the glycolytic flux in exponentially growing cells of L. lactis MG1363. Introduction of an uncoupled ATPase activity results in uncoupling of glycolysis from biomass production. With MG1363 growing in defined medium supplemented with glucose, the ATP demanding processes do not have a significant control on the glycolytic flux; it appears that glycolysis is running at maximal rate. It is likely that the flux control is distributed over many enzymes in L. lactis, but it cannot yet be excluded that one of the remaining glycolytic steps is a rate-limiting step for the glycolytic flux.

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst.

Abstract: An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5′-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of α2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and α2,3-sialyltransferase. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 516–524, 2002.

Plant Pyruvate Kinase

Abstract: Pyruvate kinase is an important enzyme of glycolytic pathway that also functions in providing carbon skeleton for fatty acid biosynthesis. It has been purified to near homogeneity from Ricinus communis, Selenastrum minutum, Cynodon dactylon, Brassica campestris and B. napus, and characterised. Partially purified preparations are reported from several other sources. A phosphoenolpyruvate (PEP) phosphatase accompanies pyruvate kinase. In plants, two isozymes of pyruvate kinase are reported, namely cytosolic and plastidic. Isoforms of cytosolic pyruvate kinase have also been reported from spinach. In most cases pyruvate kinase is a tetrameric protein and the molecular mass lies between 200 to 250 kDa. The pH optimum is in the range of 6.2 to 7.5. It requires both Mg2+ and K+ for maximum activity. ATP, citrate, and oxalate inhibit pyruvate kinase in most cases. A sequential compulsory ordered mechanism of binding of substrates to the enzyme has been proposed.

Proteomic evaluation of intermediary metabolism enzyme proteins in fetal Down's syndrome cerebral cortex.

Abstract: Trisomy 21 (Down's syndrome) is the most common genetic cause of human mental retardation. In Down's syndrome (DS) patients, deteriorated glucose, lipid, purine, folate and methionine/homocysteine metabolism has been reported. In our study, we used a proteomic approach to evaluate protein expression of enzyme proteins of intermediary metabolism in the brain of Down's syndrome fetuses. In fetal DS brain, we detected increased protein levels of mitochondrial aconitase as well as NADP-linked isocitrate dehydrogenase, decreased protein expression of citrate synthase and cytosolic aspartate aminotransferase. From two spots that corresponded to either pyruvate kinase M1 or M2 isozymes, significant elevation was observed only in one, while the second spot as well as the sum of the spots showed no differences between DS and controls. These results suggest derangement of intermediary metabolism during prenatal development of DS individuals.

Karyopherin alpha2: a control step of glucose-sensitive gene expression in hepatic cells.

Abstract: Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin alpha2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin alpha2, not alpha1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin alpha2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein-karyopherin alpha2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin alpha2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin alpha2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin alpha2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin alpha2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

Pyruvate Site of Pyruvate Phosphate Dikinase: Crystal Structure of the Enzyme−Phosphonopyruvate Complex, and Mutant Analysis†,‡

Abstract: Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, phosphonopyruvate (Ki = 3 μM), have been obtained in the presence of Mg2+. The structure has been determined and refined at 2.2 A resolution, revealing that the Mg2+-bound phosphonopyruvate binds in the α/β-barrel's central channel, at the C-termini of the β-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK−phosphonopyruvate complex and the structures of two complexes of pyruvate kinase, one with Mg2+-bound phospholactate and the other with Mg2+-oxalate and ATP, revealed that the two enzymes share some key features that facilitate commo...

Regulation of fermentation in a ruminal bacterium, Streptococcus bovis, with special reference to rumen acidosis

Abstract: When high-starch diets are fed to ruminants, Streptococcus bovis often predominates in the rumen and produces lactate as the major fermentation product. Lactate production by S. bovis is regulated by the activity ratio of lactate dehydrogenase (LDH) to pyruvate formate-lyase (PFL) in response to an energy supply or the intracellular pH. The activities of LDH and PFL are affected not only by the amounts of enzyme proteins, but also by the concentrations of allosteric effectors, such as fructose-1, 6-bisphosphate (FBP) and triose phosphates. Synthesis of LDH and PFL is regulated at the transcriptional level in opposing directions. The inverse relationship between LDH and PFL synthesis amplifies a change in the proportion of lactate in the total fermentation products. A high capacity to regulate lactate production in response to low pH allows S. bovis to grow at low pH and as one of the most acid-tolerant bacteria among the prevailing ruminal bacteria, S. bovis also has a high capacity to extrude intracellular H+ by an F1F0 H+-ATPase, which is important for acid tolerance. In addition, S. bovis is able to augment H+-ATPase synthesis at the transcriptional level in response to low pH. The activities of H+-ATPase and pyruvate kinase are enhanced by FBP and strongly inhibited by inorganic phosphate (Pi). Intracellular FBP and Pi concentrations affect sugar metabolism in S. bovis through regulation of the expression of catabolic genes and controlling the activity of sugar transport systems. Synthesis of the enzymes involved in sugar transport and fermentation appears to be regulated by the catabolite control protein A (CcpA) –phosphotransferase system (PTS) system.

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP‐utilizing enzymes

Abstract: The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).

Alanine prevents the reduction of pyruvate kinase activity in brain cortex of rats subjected to chemically induced hyperphenylalaninemia.

Abstract: The mechanisms by which phenylalanine is toxic to the brain in phenylketonuria are not fully understood. Considering that brain glucose metabolism is reduced in these patients, our main objective was to determine pyruvate kinase activity in brain cortex of rats subjected to acute and chronic chemically induced hyperphenylalaninemia. The effect of alanine administration on the enzyme activity in the treated rats was also investigated. We also studied the in vitro effect of the two amino acids on pyruvate kinase activity in brain cortex of nontreated rats. The results indicated that phenylalanine inhibits pyruvate kinase in vitro and in vivo and that alanine prevents the inhibitory effect of phenylalanine on the enzyme activity. Considering the crucial role pyruvate kinase plays in glucose metabolism in brain, it is possible that inhibition of this enzyme activity may contribute to the brain damage characteristic of this disease.

Pyruvate-kinase as a novel target molecule

Abstract: The present invention relates to the use a pyruvate-kinase or a nucleic acid coding therefor as a target for the modulation of apoptotic processes, particularly as a target molecule for the diagnosis, prevention or treatment of apoptosis-associated disorders, such as tumours

Enzymic routes to dihydroxyacetone phosphate or immediate precursors

Abstract: Dihydroxyacetone phosphate (DHAP) is an important substrate for enzymic syntheses of saccharides by means of dihydroxyacetone phosphate aldolases. A convenient precursor of DHAP is L-glycerol-3-phosphate (L-G3P), the enzymic synthesis of which may be achieved by phosphate transfer owing to an ATP-regenerating system. The phosphorylation of both glycerol and dihydroxyacetone by means of an ATP-regenerating system based on phosphoenolpyruvate, pyruvate kinase and,glycerol kinase is examined in order to optimize the reaction conditions. The composition of the reaction mixture should not exceed concentrations of 300 mM of each phosphoenolpyruvate and glycerol. Otherwise, the productivity would be affected by substrate inhibition. An operating temperature of 50 degreesC may be applied, when the enzymes should be used only once. At a temperature of 35 degreesC, a high half-life of both enzymes glycerol kinase and pyruvate kinase is achieved. Various concentrations of the enzymes are tested to optimize the use of the enzymatic activity. A ratio of 2:3 of pyruvate kinase and glycerol kinase,activity seems to be adequate. Further studies show that 10 mM ATP is enough to maintain a permanent supply of the cofactor. It is shown, how DHAP may be obtained from L-G3P by means of an L-G3P oxidase and catalase, the latter being used not only to decompose the hydrogen peroxide formed during the course of reaction, but also to supply the reaction system with oxygen. This is achieved by feeding a H2O2 Solution in a way to keep the oxygen level constant in the reaction mixture. (C) 2002 Elsevier Science B.V. All rights reserved.