Showing papers on "Pyruvate dehydrogenase kinase published in 1997"

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

Abstract: During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.

Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate.

Abstract: We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.

Treatment of congenital lactic acidosis with dichloroacetate

Abstract: The term congenital lactic acidosis (CLA) refers to a group of inborn errors of mitochondrial metabolism variably characterised by progressive neuromuscular deterioration and accumulation of lactate and hydrogen ions in blood, urine and/or cerebrospinal fluid, frequently resulting in early death.1-4 The incidence and prevalence of CLA are unknown, although it has been estimated that there are approximately 250 new cases recognised in the US per year (personal communication). Thus, with an estimated annual mortality attrition rate of 20%, at least 1000 cases exist in the general US population. Recent diagnostic advances have allowed the biochemical or molecular identification of specific enzyme defects in the majority of infants and children with CLA. Most identifiable cases involve inherited or spontaneous mutations in the pyruvate dehydrogenase complex (PDC) or in one or more enzymes of the respiratory chain.2 4 A few cases have been reported that involve deficiencies in enzymes of the tricarboxylic acid cycle, such as fumarase, or of gluconeogenesis, such as pyruvate carboxylase (PC) or phosphoenolpyruvate carboxykinase (PEPCK). In a substantial number of patients, however, the specific biochemical defect fails to be determined by established techniques. Hyperlactataemia is the defining biochemical abnormality in children with CLA and, in the absence of hypoxia, should be considered a surrogate marker for underlying failure of mitochondrial energy metabolism.5 This concept is most readily appreciated by considering mitochondrial enzyme deficiencies. Figure 1 summarises the major steps of carbohydrate oxidation in mammalian cells. Note that the oxidative fate of pyruvate is to be irreversibly decarboxylated to acetyl CoA. This reaction is catalysed by PDC, a series of linked enzymes located in the inner mitochondrial membrane (fig 2). Under aerobic conditions, the activity of PDC determines the rate at which all cells oxidise glucose, pyruvate, and lactate. Figure 1 Pathways of pyruvate metabolism and oxidative phosphorylation. …

Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess

Abstract: In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.

Substrate Kinetics of the Plant Mitochondrial Alternative Oxidase and the Effects of Pyruvate.

Abstract: The kinetics of alternative oxidase (AOX) of Arum italicum spadices and soybean (Glycine max L.) cotyledons were studied both with intact mitochondria and with a solubilized, partially purified enzyme. Ubiquinone analogs were screened for their suitability as substrates and ubiquinol-1 was found to be most suitable. The kinetics of ubiquinol-1 oxidation via AOX in both systems followed Michaelis-Menten kinetics, suggesting that the reaction is limited by a single-step substrate reaction. The kinetics are quite different from those previously described, in which the redox state of ubiquinone-10 was monitored and an increase in substrate was accompanied by a decrease in product. The difference between the systems is discussed. Pyruvate is a potent activator of the enzyme and its presence is essential for maximum activity. The addition of pyruvate to the solubilized enzyme increased the maximum initial velocity from 6.2 [plus or minus] 1.3 to 16.9 [plus or minus] 2.8 [mu]mol O2 mg-1 protein min-1 but had little effect on the Michaelis constant for ubiquinol-1, an analog of ubiquinol, which changed from 116 [plus or minus] 73 to 157 [plus or minus] 68 [mu]M. It is concluded that pyruvate (and presumably other keto acids) increases the activity of AOX but does not increase its affinity for its substrate. In agreement with this is the finding that removal of pyruvate (using lactate dehydrogenase and NADH) leads to an 80 to 90% decrease in the reaction rate, suggesting that pyruvate is important in the mechanism of reaction of AOX. The removal of pyruvate from the enzyme required turnover, suggesting that pyruvate is bound to the enzyme and is released during turnover.

Role of the stimulatory proteins sp1 and sp3 in the regulation of transcription of the rat pyruvate kinase m gene

Abstract: Site-directed mutagenesis of cis-regulatory elements in the 5' flanking region of the rat pyruvate kinase M gene revealed that two out of the three GC boxes (-133/-124 and -48/-39) are involved in the stimulation of a core promoter (-35/+46). These two regions were also protected in DNaseI footprinting assays. Sp1 and Sp3 were identified as binding proteins to all three GC boxes by supershift experiments. Cotransfections in Drosophila SL2 cells revealed a strong stimulatory function of Sp1 and a synergistic effect of Sp3 to Sp1 in the activation of the pyruvate kinase M promoter. No inhibitory effect of Sp3 was detected. These data indicate that binding of Sp1 at two GC boxes is required for full promoter activity of the pyruvate kinase M gene and thus contributes to the observed cell-cycle-dependent expression of this enzyme in proliferating rat thymocytes.

Inhibition of 11β-Hydroxysteroid Dehydrogenase Type 1

Abstract: (1997). Inhibition of 11β-Hydroxysteroid Dehydrogenase Type 1. Emerging Therapeutic Targets: Vol. 1, No. 1, pp. 223-227.

Tumor-specific pyruvate kinase isoenzyme M2 involved in biochemical strategy of energy generation in neoplastic cells

Abstract: The differences in properties of pyruvate kinase (EC 2.1.7.40) from normal tissues and animal or human tumors are described and their significance for various metabolic abnormalities is reviewed. The tumor variant gamma3 from M2 isoenzyme of pyruvate kinase sensitive to L-cysteine inhibition, when over-expressed, can be used as a marker of tumorigenic transformation. It seems that this variant represents a tumor-specific oncoprotein, involved in a novel metabolic strategy of energy generation during increased cell proliferation.

Glucose transport and glucose transporter GLUT4 are regulated by product(s) of intermediary metabolism in cardiomyocytes

Abstract: Alternative substrates of energy metabolism are thought to contribute to the impairment of heart and muscle glucose utilization in insulin-resistant states. We have investigated the acute effects of substrates in isolated rat cardiomyocytes. Exposure to lactate, pyruvate, propionate, acetate, palmitate, beta-hydroxybutyrate or alpha-oxoglutarate led to the depression of glucose transport by up to 50%, with lactate, pyruvate and propionate being the most potent agents. The percentage inhibition was greater in cardiomyocytes in which glucose transport was stimulated with the alpha-adrenergic agonist phenylephrine or with a submaximal insulin concentration than in basal or fully insulin-stimulated cells. Cardiomyocytes from fasted or diabetic rats displayed a similar sensitivity to substrates as did cells from control animals. On the other hand, the amination product of pyruvate (alanine), as well as valine and the aminotransferase inhibitors cycloserine and amino-oxyacetate, stimulated glucose transport about 2-fold. In addition, the effect of pyruvate was counteracted by cycloserine. Since reversible transamination reactions are known to affect the pool size of the citrate cycle, the influence of substrates, amino acids and aminotransferase inhibitors on citrate, malate and glutamate content was examined. A significant negative correlation was found between alterations in glucose transport and the levels of citrate (P < 0.01) or malate (P < 0.01), and there was a positive correlation between glucose transport and glutamate levels (P < 0.05). In contrast, there was no correlation with changes in [1-(14)C]pyruvate oxidation or in glucose-6-phosphate levels. Finally, pyruvate decreased the abundance of GLUT4 glucose transporters at the surface of phenylephrine- or insulin-stimulated cells by 34% and 27 % respectively, as determined by using the selective photoaffinity label [3H]ATB-BMPA [[3H]2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-man nos-4-yloxy)propyl-2-amine]. In conclusion, cardiomyocyte glucose transport is subject to counter-regulation by alternative substrates. The glucose transport system appears to be controlled by (a) compound(s) of intermediary metabolism (other than glucose 6-phosphate), but in a different way than pyruvate dehydrogenase. Transport inhibition eventually occurs via a decrease in the amount of glucose transporters in the plasma membrane.

Protein engineering tests of a homology model of Plasmodium falciparum lactate dehydrogenase.

Abstract: This paper describes the testing of a homology model of Plasmodium falciparum lactate dehydrogenase (pfLDH) by protein engineering. The model had been validated in structural terms. It suggests explanations of the unusual properties of pfLDH (compared with all other LDHs). Fig. 1. Catalytic cycle for LDH, including the inhibitory complex enzyme‐ These unusual features are a lack of substrate inhibition, NAD1‐pyruvate. E, enzyme; R, NADH; O, NAD 1; P, pyruvate; L, lactate. high activity with the synthetic coenzyme 3-acetylpyridine adenine dinucleotide (APAD 1 ) and changes in residues at previously conserved positions. pfLDH shows several amino into lactate by LDH so as to regenerate NAD 1 for the acid insertions and deletions in an alignment with protein continuation of anaerobic glycolysis. This aspect of the plassequences from all other known LDHs. The most notable modial lifestyle required the evolution of an LDH enzyme that is a five amino acid insertion into the active-site loop. In would not be inhibited by high pyruvate or lactate levels—a addition, a conserved serine at position 163 is replaced by feature not so pronounced in LDH enzymes of other origins leucine. The results showed that when the unique pfLDH where pyruvate continues into the aerobic phase of metabolism. structural features were engineered into Bacillus stearo- It has been known for some considerable time that abortive thermophilus lactate dehydrogenase, the thermophilic ternary complex formation is, at least in part, responsible for enzyme acquired the properties previously uniquely asso- substrate inhibition in dehydrogenase enzymes (Fromm, 1961; ciated with the malarial enzyme. We conclude that the Gutfreund et al., 1968; Coulson and Rabin, 1969; Deng et al., homology model of the malarial enzyme is adequate for 1994). Abortive complexes in the LDH mechanism are formed the prediction of successful redesigns and, in the regions when enzyme with oxidized coenzyme still bound collides tested, is accurate. with the oxidized form of substrate in a non-productive

Mck1, a member of the glycogen synthase kinase 3 family of protein kinases, is a negative regulator of pyruvate kinase in the yeast Saccharomyces cerevisiae.

Abstract: An interaction between the Saccharomyces cerevisiae protein kinase Mck1 and pyruvate kinase (Pyk1) was detected by using the two-hybrid method. Purified Mck1 was able to phosphorylate purified Pyk1 on Ser in vitro. Pyruvate kinase activity was elevated in mck1 delta cells. Several of the phenotypes of mck1 delta mutants are similar to those observed in cells overexpressing PYK1. Co-overexpression of MCK1 suppressed all of the phenotypes associated with PYK1 overexpression. These results indicate that Mck1 negatively regulates pyruvate kinase activity, possibly by direct phosphorylation.

2-oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: Synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex

Abstract: A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphospha...

Molecular Mechanism of Regulation of the Pyruvate Dehydrogenase Complex from E. coli

Abstract: The pyruvate dehydrogenase multienzyme complex from E. coli shows a sigmoidal dependency of the reaction rate on the substrate concentration when product formation is followed in the presence of physiological concentrations of the cofactor thiamin diphosphate. To elucidate the molecular mechanism of this regulation, the influence of the substrate pyruvate on the coenzyme−protein interaction has been investigated using several coenzyme analogues. The observed binding constants of all coenzymatically active analogues are increased in the presence of the substrate pyruvate, whereas those of all coenzymatically inactive analogues are not altered in the presence of pyruvate. This points to an increased binding affinity of a reaction-intermediate−coenzyme complex to the protein. Since cofactor binding and dissociation at physiological concentrations of thiamin diphosphate are slow compared to the catalytic reaction, a slow transition to the active state of the enzyme occurs. After lowering the pyruvate concentr...

Effect of cyclic polylactates on tumor cells and tumor bearing mice

Abstract: We studied the effect of cyclic polylactates ranging in size from a degree of polymerization number of 3 to 13 on pyruvate kinase, lactic dehydrogenase, anaerobic glycolysis, growth of tumor cells and survival of tumor bearing mice. Pyruvate kinase and lactic dehydrogenase activities were both inhibited by cyclic polylactates, and the inhibition mechanism of cyclic polylactates on lactic dehydrogenase was noncompetitive. About half the anaerobic glycolytic activity of FM3A ascites tumor cells was inhibited and tumor cell growth was also effectively inhibited by cyclic polylactates. Mice, which were treated with cyclic polylactates after inoculation of FM3A ascites tumor cells lived significantly longer than mice, which were treated with vehicle or non mice.

Expression and Characterisation of the Homodimeric E1 Component of the Azotobacter vinelandii Pyruvate Dehydrogenase Complex

Abstract: We have cloned and sequenced the gene encoding the homodimeric pyruvate dehydrogenase component (Elp) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and expressed and purified the E1p component in Escherichia coli. Cloned E1p can be used to fully reconstitute complex activity. The enzyme was stable in high ionic strength buffers, but was irreversibly inactivated when incubated at high pH, which presumably was caused by its inability to redimerize correctly. This explains the previously found low stability of the wild-type E1p component after resolution from the complex at high pH. Cloned E1p showed a kinetic behaviour exactly like the wild-type complex-bound enzyme with respect to its substrate (pyruvate), its allosteric properties, and its effectors. These experiments show that acetyl coenzyme A acts as a feedback inhibitor by binding to the E1p component. Limited proteolysis experiments showed that the N-terminal region of E1p was easily removed. The resulting protein fragment was still active with artificial electron acceptors but had lost its ability to bind to the core component (E2p) and thus reconstitute complex activity. E1p was protected against proteolysis by E2p. The allosteric effector pyruvate changed E1p into a conformation that is more resistant to proteolysis.