Showing papers on "Pyruvate dehydrogenase kinase published in 1993"
TL;DR: Mitochondria from soybean shoots displayed high alternative oxidase activity with succinate and malate as substrates but lower activity with exogenous NADH; addition of pyruvate stimulated the activity with NADH up to that seen with succinates.
272 citations
TL;DR: In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/pyruvate, fructose, alanine or glutamine, following metformin addition is observed, and this could explain the stimulation of pyruVate-kinase activity following met formin addition and thus the inhibition of gluconeogenesis.
Abstract: Metformin (dimethylbiguanide) has been used for more than 30 years as an antihyperglycemic agent in the treatment of diabetes mellitus, but its effect on gluconeogenesis is still controversial. In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/pyruvate (10:1, mol/mol), fructose, alanine or glutamine, following metformin addition, is observed. Moreover, in hepatocytes perifused with dihydroxyacetone as the gluconeogenic substrate and treated with 0.5 mM metformin, an inhibition of the glucose flux and a simultaneous stimulation of the lactate/pyruvate flux were observed. This enhancement of lactate/pyruvate formation appears to be due to an effect on the pyruvate-kinase enzyme. A direct effect of metformin on pyruvate kinase cannot explain this result, since pyruvate-kinase activity was not affected by metformin at this concentration. In contrast, the addition of metformin caused a significant decrease in the cellular ATP concentration, a known allosteric inhibitor of this enzyme. This could explain the stimulation of pyruvate-kinase activity following metformin addition and thus the inhibition of gluconeogenesis.
149 citations
TL;DR: Molecular cloning of pyruvate dehydrogenase kinase establishes a new eukaryotic family of protein kinases that is related to a prokaryoticFamily ofprotein kinases.
146 citations
TL;DR: The L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene in the endocrine beta-cell type and is shown to increase 4-fold at maximal stimulation, suggesting that both transcriptional and post-transcriptional effects contribute to mRNA accumulation.
122 citations
TL;DR: To maximize catalytic efficiency in metabolic pathways of both prokaryotic and eUkaryotic cells, enzymatic components are occasionally clustered or complexed physically.
Abstract: To maximize catalytic efficiency in metabolic pathways of both prokaryotic and eUkaryotic cells, enzymatic components are occasionally clustered or complexed physically. Organization of multiple catalytic functions into a single enzyme complex can be accomplished in two ways. First, multi functional enzymes utilize a single polypeptide chain to catalyze more than one enzymatic function; an example of a multifunctional enzyme is the mammalian fatty'acid synthase. A more common means for clustering enzymatic functions is the assembly of mUltiple catalytic components into complexes held together by noncovalent bonds. The most prominent examples of multienzyme complexes are the a-keto acid dehydrogenase multienzyme complexes that catalyze the oxidative decarboxylation of pyruvate, a-keto-
118 citations
TL;DR: The mutant dihydrolipoamide dehydrogenase displayed high levels of activity with this coenzyme, the values of Km, kcat, and kcat/Km comparing favorably with those found for the wild-type enzyme operating with NAD.
Abstract: Systematic replacement of a set of amino acids in the beta alpha beta-fold of the NAD-binding domain of Escherichia coli dihydrolipoamide dehydrogenase has been used to convert its coenzyme specificity from NAD to NADP. After comparison with the homologous enzyme glutathione reductase, Glu 203 was replaced with a valine residue, thereby eliminating the potential to form hydrogen bonds with the 2'- and 3'-OH groups of the adenine ribose in NAD. Similarly, Met 204, Pro 210, Phe 205, and Asp 206 were replaced by an arginine, an arginine, a lysine, and a histidine residue, respectively, to provide a nest of positive charge to accommodate the 2'-phosphate group of the incoming NADP. In addition, Gly 185 and Gly 189 in the beta alpha beta motif were replaced with alanine residues to facilitate the positioning of the newly introduced Val 203 by allowing a flip of the peptide bond between residues Gly 180 and Gly 181. Wild-type dihydrolipoamide dehydrogenase is inactive with NADP, but the mutant enzyme displayed high levels of activity with this coenzyme, the values of Km, kcat, and kcat/Km comparing favorably with those found for the wild-type enzyme operating with NAD. The mutant enzyme was also capable of assembly in vitro to form an active pyruvate dehydrogenase multienzyme complex, the coenzyme specificity of which reflected that of its dihydrolipoamide dehydrogenase component. These experiments should make it possible now to study the effects in vivo of requiring a crucial catabolic enzyme to function with the wrong coenzyme, an important extension of protein engineering into the living cell.
105 citations
TL;DR: Kinetic studies showed that the rate of radical formation was independent of ionic strength and the Km's for SAM and inactive PFL were determined to be 2.8 and 1.2 microM, respectively, indicating that the binding site for SAM resides on AE.
Abstract: Pyruvate formate-lyase is a radical-containing enzyme that catalyzes the nonoxidative cleavage of pyruvate via a postulated homolytic mechanism. The formation of this enzymic radical in vitro requires an activating system composed of PFL-activating enzyme, S-adenosylmethionine, ferrous ion, a reduced flavin, DTT, and pyruvate as an allosteric effector. The need for large quantities of PFL-activating enzyme for biochemical and biophysical studies on the mechanism of protein radical formation has prompted us to clone the act gene and overexpress the gene product in Escherichia coli. Using PCR technology, the act gene was isolated and subcloned into various expression vectors. The overexpression of the protein was as high as 30-50% of the total cellular protein. However, the majority of the protein resided in the form of insoluble inclusion bodies. A procedure was developed to denature and isolate the inclusion bodies followed by refolding under anaerobic conditions. This purification method affords 5 mg of purified protein from 1 g of cells. Biochemical characterization demonstrated that the enzyme can bind one Fe(II) per protein monomer, and the protein did not exhibit any visible chromophore as previously observed. Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometries. Kinetic studies showed that the rate of radical formation was independent of ionic strength and the Km's for SAM and inactive PFL were determined to be 2.8 and 1.2 microM, respectively. Fluorescent binding data revealed that the Kd for SAM binding to the activating enzyme alone was comparable to the Km for SAM in the PFL activation indicating that the binding site for SAM resides on AE.(ABSTRACT TRUNCATED AT 250 WORDS)
95 citations
TL;DR: The regulatory gene PDC2 was identified in a screen for mutations affecting pyruvate decarboxylase activity in yeast and cloned and sequenced this gene, which is either important for a high basal level of PDC gene expression or it plays a positive role in the autoregulation that controls expression of P DC1 and PDC5.
Abstract: The regulatory gene PDC2 was identified in a screen for mutations affecting pyruvate decarboxylase activity in yeast. I have cloned and sequenced this gene. The predicted protein of 925 amino acids has no homology to any sequence in the databases. However, the protein sequence is rich in asparagine and serine residues, as is often found for transcriptional regulators. The PDC2 deletion mutant exhibits a phenotype very similar to, but more severe than that of the point mutant: a strongly reduced pyruvate decarboxylase specific activity, slow, respiration-dependent growth on glucose, and accumulation of pyruvate. The activity of other glycolytic enzymes seems to be unaffected by the pdc2Δ mutation. Synthesis of pyruvate decarboxylase is regulated by PDC2 at the transcriptional level. Expression of the major structural gene for pyruvate decarboxylase, PDC1, is strongly reduced in pdc2Δ mutants. Transcription of the generally more weakly expressed PDC5 gene appears to be entirely abolished. However, glucose induction of pyruvate decarboxylase synthesis is unaffected. Thus, PDC2 is either important for a high basal level of PDC gene expression or it plays a positive role in the autoregulation that controls expression of PDC1 and PDC5.
90 citations
TL;DR: Experiments reported here provide the first direct evidence that pyruvate release in African trypanosomes is catalyzed by a specific transport system and implicate this transporter as a vehicle for delivering toxic alkylating agents intotrypanosomal cells.
57 citations
TL;DR: The results indicate that relatively equal amounts of pyruvate enter the citric acid cycle by carboxylation and decarboxylations in pancreatic islets.
Abstract: The routes by which glucose-derived pyruvate is metabolized to enter mitochondrial pathways to stimulate insulin release in the pancreatic β cell are unknown. The 14 CO 2 ratios assay was used to estimate the fractions of glucose-derived pyruvate that enter the citric acid cycle by carboxylation and decarboxylation in pancreatic islets. Pyruvate 14 CO 2 ratios were estimated with glucose, and acetate 14 CO 2 ratios were estimated with succinate methyl ester. Glucose and methyl succinate, which are insulin secretagogues, gave more accurate ratios than ratios estimated with pyruvate and acetate, which do not initiate insulin release. The results indicate that relatively equal amounts of pyruvate enter the citric acid cycle by carboxylation and decarboxylation.
51 citations
TL;DR: The homology of the sequence showed that the gene encodes phosphofructokinase, which was expressed in E. coli cells, and the evidence suggesting that both genes constitute an operon is presented.
Abstract: Pyruvate kinase from Bacillus stearothermophilus is an allosteric enzyme activated by AMP or ribose 5-phosphate but not by fructose 1,6-bisphosphate. The gene for the enzyme was cloned in Escherichia coli and its entire nucleotide sequence was determined. The deduced amino acid sequence consisted of 587 residues and the molecular mass was calculated to be 62 317 Da. The sequence was highly similar to other pyruvate kinases, indicating that they have the same evolutional origin. Similarly to the E. coli enzymes, the enzyme does not contain an N-terminal domain, in contrast to the eukaryotic pyruvate kinases. However, the Bacillus stearothermophilus enzyme had an extra C-terminal sequence consisting of about 110 amino acid residues. A phosphoenolpyruvatebinding motif, which is observed in pyruvate phosphate dikinase, phosphoenolpyruvate: sugar phosphotransferase system enzyme I and phosphoenolpyruvate synthase, was present in the extra C-terminal sequence. There was an open reading frame upstream of the pyruvate kinase gene. The homology of the sequence showed that the gene encodes phosphofructokinase. Both phosphofructokinase and pyruvate kinase were expressed in E. coli cells, and the evidence suggesting that both genes consitute an operon is presented.
TL;DR: Acetate, when added to islets maintained at low glucose, increased dimethyl succinate metabolism to almost that of islet maintained at high glucose, consistent with a low amount of pyruvate dehydrogenase being unable to supply acetyl-CoA for condensation with oxalacetate and that the rate of the citric acid cycle could be enhanced by adding acetate which can bypass the reaction catalyzed by pyruVate dehydration.
TL;DR: The provision of the high‐fat diet for 28 days evoked a significant decline in cardiac PDHa, activity, together with marked increases in the activity of PDH kinase measured in isolated mitochondria and freshly‐prepared cardiomyocytes from adult rats.
TL;DR: It is shown that a hydrophobic region of the β‐subunit of the PDH E1 αβ enzymes likely contains a binding site for the thiazolium ring of TPP and key motifs are retained in common by all the TPP‐dependent enzymes considered, which are essential for catalysis.
TL;DR: The effects of phenylethylbiguanide (phenformin) on the rate of gluconeogenesis and metabolite profiles in the perfused liver are similar to those caused by DCMU, supporting a mitochondrial locus of action for this hypoglycaemic agent.
TL;DR: The determination of the three-dimensional structures of transketolase, pyruvate oxidase and pyruVate decarboxylase has revealed a common thiamin-binding fold and provided the first structural insights into enzymaticThiamin catalysis.
TL;DR: The lipoyl prosthetic group on one of the twolipoyl domains of E2 subunits is critically important for maintaining E2-activated kinase function and contributes to binding of the kinase to E2.
TL;DR: Mitochondrial pyruvate-H+ symport was the normal, highly efficient (rather than controlling) mechanism for pyruVate entry into the mitochondria where PDH regulation controlled pyruviate oxidation.
TL;DR: Treatment of 18 h-starved rats with dexamethasone and subsequent isolation and incubation of the hepatocytes in the presence of the steroid increased gluconeogenic flux with both pyruvate kinase flux and glucose synthesis with both substrates employed, indicating that the steroid had no effect on the partitioning of phosphoenolpyruvates between pyruVate and lactate formation and gluconeogenesis.
Abstract: Treatment of 18 h-starved rats with dexamethasone and subsequent isolation and incubation of the hepatocytes in the presence of the steroid increased gluconeogenic flux with both 1.0 mM pyruvate and 1.0 mM lactate plus 0.2 mM pyruvate as the substrate. The magnitude of stimulation was comparable with both substrates. The increase in glucose output was accompanied by an increased flux through pyruvate carboxylase, although the absolute flux and magnitude were considerably less in the presence of the more reduced substrate. The effect of the steroid on the flux through pyruvate dehydrogenase was substrate-dependent, an inhibition occurring with the more oxidized substrate. There was no effect of steroid treatment on [1-14C]lactate or pyruvate oxidation or on tricarboxylic-acid-cycle flux as measured by [3-14C]pyruvate oxidation. Dexamethasone treatment resulted in a parallel increase in both pyruvate kinase flux and glucose synthesis with both substrates employed, indicating that the steroid had no effect on the partitioning of phosphoenolpyruvate between pyruvate and lactate formation and gluconeogenesis. Similarly there was no effect of the steroid on either the activity ratio or the total pyruvate kinase activity in the cells. It is suggested that the acute effect of the dexamethasone to increase gluconeogenesis resides at the level of phosphoenolpyruvate formation, i.e. pyruvate carboxylase and possibly phosphoenolpyruvate carboxykinase.
TL;DR: Evidence is provided in support of the concept that selective suppression of pyruvate oxidation in oxidative muscles during early starvation and during the initial phase of refeeding is achieved because of differential sensitivity of glucose uptake/phosphorylation and pyruVate oxidation to lipid-fuel utilization.
TL;DR: The specific activity of 13-HODE dehydrogenase shows a strong positive correlation with the degree of differentiation of intestinal mucosal cells from both the small and large intestines, raising the possibility the enzyme plays a role in the process of cellular differentiation.
TL;DR: Pyruvate, which protects the heart by inducing a large recovery of the contractile activity after ischemia, markedly prevents the loss of glutathione, and total sulfhydryl groups of mitochondria do not undergo significant variations upon ischemIA and reperfusion in the presence of pyruvates.
Abstract: Ischemia or hypoxia followed by reperfusion determine a large release of glutathione from isolated and perfused rat heart. The effects of glucose and/or pyruvate administered during ischemia/reperfusion or hypoxia/reperfusion on the release of cytosolic and mitochondrial glutathione are compared. During ischemia, mitochondrial glutathione is released from the mitochondrion to the cytosol forming a unique pool that leaks out to the interstitial space. Reperfusion causes a large release of total glutathione, particularly from cytosol. Total sulfhydryl groups do not undergo modifications after ischemia, while they appear to decrease upon reperfusion. Pyruvate, which protects the heart by inducing a large recovery of the contractile activity after ischemia, markedly prevents the loss of glutathione. Also total sulfhydryl groups of mitochondria do not undergo significant variations upon ischemia and reperfusion in the presence of pyruvate. During hypoxia, in the absence of glucose, glutathione is mainly lost from the cytosol, while the mitochondrial pool appears to be preserved; in hypoxia, at variance with the ischemic conditions, pyruvate does not show any beneficial effect. The action of pyruvate appears to be multifactorial and its effects are discussed by considering its action on the hydrogen peroxide breakdown, protection of pyruvate dehydrogenase, anaerobic production of ATP and diminution of the intracellular concentration of inorganic phosphate.
01 Jan 1993
TL;DR: Comparison of total PDHC activity with [1-14C]-pyruvate oxidation rates, both measured in the supernatant prepared from fresh muscle, shows an equimolar correlation, indicating that totalPDHC activity is rate limiting in the assay for the pyruvates oxidation rate.
Abstract: A sensitive radiochemical method for the determination of the pyruvate dehydrogenase complex (PDHC) activity in skeletal muscle tissue, based on the decarboxylation of [1-14C]-pyruvate to 14CO2, is described. Measurements can be carried out either in muscle homogenate or in 600-g supernatant, both obtainable from a small muscle biopsy specimen (20 mg). In addition to NAD+, thiamine pyrophosphate and coenzyme A in the incubation mixture, a preparation of NADH:cytochrome c reductase (NADHCR) together with cytochrome c has a stimulating effect on the PDHC activity. NADHCR constitutes an oxidation system for NADH to prevent feedback inhibition. Addition of L-carnitine also results in stimulation of PDHC by trapping the produced acetyl-CoA as acetylcarnitine. Special care for radioactive pyruvate, with freeze drying and storage at -20 degrees C under nitrogen, and determination of the purity during every PDHC assay, is required. In the presented assay a Km value of 0.084 mmol/l was found for pyruvate. Nonsigmoidal kinetics was found with a Hill coefficient of 1.63. With the described method, a totally Mg2+,Ca(2+)-stimulated PDHC activity is measured. Addition of a purified specific pyruvate dehydrogenase phosphatase did not yield a higher PDHC activity. Finally, comparison of total PDHC activity with [1-14C]-pyruvate oxidation rates, both measured in the supernatant prepared from fresh muscle, shows an equimolar correlation, indicating that total PDHC activity is rate limiting in the assay for the pyruvate oxidation rate. Neonatal muscle exhibits five to ten times lower PDHC activities and pyruvate oxidation rates than controls (age > 3 years).
TL;DR: It is concluded that plant cytosolic pyruvate kinase activity and regulation is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme.
Abstract: Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo.
TL;DR: Binding of the kinase to the lipoyl domain region elicits a structural change which enhances kinase activity; however, other processes are required to explain the very large enhancement in phosphorylation of E1 effected by intact E2 core.
TL;DR: The results are consistent with the hypothesis that activation of the PDH complex immediately following insulin administration is not mediated by a decreased PDH kinase activity, however, with daily insulin therapy in diabetes,activation of thePDH complex results from decreased PDh kinaseactivity.
Abstract: The pyruvate dehydrogenase (PDH) complex undergoes reversible phosphorylation catalyzed by a PDH kinase (inactivating) and a PDH phosphatase (activating). In skeletal muscle, a decreased proportion of active PDH (PDHa) complex limits glucose oxidation in insulin-deficient states. The time-course for reactivation of the PDH complex by insulin in skeletal muscle of diabetic rats is important to understanding the potential mode of the action of insulin in regulating glucose metabolism. A single injection of insulin (1 U/kg) completely reversed the effects of alloxan-diabetes on PDHa activity within 1 hour. The normalization of the effects of diabetes on PDHa activity by insulin was maintained for a minimum of 6 hours. The increase in PDHa activity occurred before an insulin-induced decrease in plasma free fatty acids levels, demonstrating a dissociation between the antilipolytic effects of insulin and its ability to activate the PDH complex. PDH kinase activity was not normalized to control values following a single injection of insulin. Therefore, acute (1 to 6 hours) insulin-mediated activation of the PDH complex does not result from a decrease in PDH kinase activity. However, longer-term insulin therapy (1 U/kg body weight; twice daily) restored both PDHa and PDH kinase activities. The results are consistent with the hypothesis that activation of the PDH complex immediately following insulin administration is not mediated by a decreased PDH kinase activity. However, with daily insulin therapy in diabetes, activation of the PDH complex results from decreased PDH kinase activity.
TL;DR: It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition.
Abstract: The regulation of the pea (Pisum sativum) leaf mitochondrial pyruvate dehydrogenase complex by respiratory rate and oxidative phosphorylation has been investigated by measuring the respiratory activity, the redox poise of the quinone pool (Q-pool), and mitochondrial pyruvate dehydrogenase (mtPDC) activity under various metabolic conditions. It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition. In the presence of ADP significant inactivation of PDC, which was sensitive to oligomycin, was observed with all substrates, apart from pyruvate, suggesting that inactivation was due to ATP formation. Inactivation of PDC by ADP addition was observed even in the presence of carboxyatractyloside, an inhibitor of the ATP/ADP translocator, suggesting that other mechanisms to facilitate the entry of adenylates, in addition to the adenylate carrier, must exist in plant mitochondria.
TL;DR: In the presence of both coupling enzymes, the rate of conversion of pyruvate to citrate was higher than predicted on the basis of the Michaelis-Menten relationship of the two coupling enzymes.
TL;DR: NH4+ was able to stimulate PDH kinase further in the presence of saturating K+.
Abstract: The pyruvate dehydrogenase kinase-catalyzed inactivation of the pyruvate dehydrogenase complex was studied using dialyzed, soluble proteins from mitochondria purified from green leaf tissue of Pisum sativum L. seedlings. At subsaturating ATP concentrations, K+ or NH4+, but not Na+, stimulated the pyruvate dehydrogenase kinase by lowering the Km(ATP). Micromolar concentrations of NH4+ were required to produce the same effect as millimolar concentrations of K+. This is apparent from the observations that the activation constant (Kact) for NH4+ was 0.1 mM, whereas the Kact(K+) was 0.7 mM. Maximal pyruvate dehydrogenase kinase velocities attained with NH4+ were higher than those with K+, and, therefore, NH4+ was able to stimulate PDH kinase further in the presence of saturating K+. This result supports our conclusion that photorespiratory NH4+ production in plant mitochondria may be involved in regulating the entry of carbon into the Krebs cycle by way of the pyruvate dehydrogenase complex.
TL;DR: The results confirm that a stimulation of pyruvate kinase activity following treatment with lipopolysaccharide is not involved in the inhibition of gluconeogenesis, but that the effect resides at the level of phosphoenolpyruvates formation.
Abstract: The effect of treatment of rats with bacterial endotoxin on gluconeogenesis and the flux through pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase and pyruvate dehydrogenase (PDH) was measured in isolated hepatocytes, prepared from animals starved for 18 h, incubated in the presence of 1 mM pyruvate. The lipopolysaccharide reduced gluconeogenesis by 50% and lowered the flux through pyruvate kinase, PEPCK and pyruvate carboxylase by comparable amounts. There was no effect of endotoxaemia on PDH flux, indicating that the lowered rate of gluconeogenesis is not the result of a redistribution of pyruvate metabolism between oxidation and carboxylation. The results confirm that a stimulation of pyruvate kinase activity following treatment with lipopolysaccharide is not involved in the inhibition of gluconeogenesis, but that the effect resides at the level of phosphoenolpyruvate formation. The most favoured mechanism for the inhibition of glucose synthesis is via an inhibition of PEPCK and subsequent feedback inhibition of pyruvate carboxylase, although a secondary effect at the level of the mitochondria and pyruvate carboxylase cannot be excluded.