Showing papers on "Pyruvate dehydrogenase kinase published in 2000"
TL;DR: The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase in vivo andMeasurements of itsk cat/K m values demonstrate that ferredoxin is ahighly efficient electron carrier in both the oxidative and reductive reactions.
198 citations
TL;DR: The findings of this study indicate that increased expression of PDK isoenzymes is an important mechanism for bringing about inactivation of the pyruvate dehydrogenase complex during starvation in many but not all tissues of the body.
185 citations
TL;DR: High-fat feeding leads to selective upregulation of PDK4 expression in slow-twitch muscle in response to high-Fat feeding in vivo, which is associated with a pronounced loss of sensitivity ofPDK activity to acute inhibition by pyruvate.
Abstract: In using Western blot analysis with antibodies raised against recombinant pyruvate dehydrogenase kinase (PDK) isoforms PDK2 and PDK4, this study demonstrates selective PDK isoform switching in specific skeletal muscle types in response to high-fat feeding that is associated with altered regulation of PDK activity by pyruvate. The administration of a diet high in saturated fats led to stable (approximately 2-fold) increases in PDK activities in both a typical slow-twitch (soleus [SOL]) muscle and a typical fast-twitch (anterior tibialis [AT]) muscle. Western blot analysis revealed that high-fat feeding significantly increased (approximately 2-fold; P < 0.001) PDK4 protein expression in SOL, with a modest (1.3-fold) increase in PDK2 protein expression. The relative increase in PDK4 protein expression in SOL was associated with a 7.6-fold increase in the pyruvate concentration that was required to elicit a 50% active pyruvate dehydrogenase complex, which indicates a marked decrease in the sensitivity of PDK to inhibition by pyruvate. In AT muscle, high-fat feeding elicited comparable (1.5- to 1.7-fold) increases (P < 0.05) in PDK4 and PDK2 protein expression. Loss of sensitivity of PDK to inhibition by pyruvate was less marked. The data suggest that a positive correlation exists between increases in PDK4 expression and the propensity with which muscles use lipid-derived fuels as respiratory substrates rather than with the degree of insulin resistance induced in skeletal muscles by high-fat feeding. In conclusion, high-fat feeding leads to selective upregulation of PDK4 expression in slow-twitch muscle in response to high-fat feeding in vivo, which is associated with a pronounced loss of sensitivity of PDK activity to acute inhibition by pyruvate. Thus, increased PDK4 expression may underlie the stable modification of the regulatory characteristics of PDK observed in slow-twitch muscle in response to high-fat feeding.
166 citations
TL;DR: A substantial influence of cytosolic NADH on cardiac O(2)(-).
Abstract: Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O2 −·). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical ...
131 citations
TL;DR: Neuronal pyruvate carboxylation was confirmed by the higher specific activity of glutamate than of glutamine after intrastriatal injection of [1-14C]pyruvates into anesthetized mice, and equilibration of malate, formed through pyruVate car boxylation, with fumarate was demonstrated.
Abstract: The cerebral metabolism of lactate was investigated. Awake mice received [3-13C]lactate or [1-13C]glucose intravenously, and brain and blood extracts were analyzed by 13C nuclear magnetic resonance spectroscopy. The cerebral uptake and metabolism of [3-13C]lactate was 50% that of [1-13C]glucose. [3-13C]Lactate was almost exclusively metabolized by neurons and hardly at all by glia, as revealed by the 13C labeling of glutamate, gamma-aminobutyric acid and glutamine. Injection of [3-13C]lactate led to extensive formation of [2-13C]lactate, which was not seen with [1-13C]glucose, nor has it been seen in previous studies with [2-13C]acetate. This formation probably reflected reversible carboxylation of [3-13C]pyruvate to malate and equilibration with fumarate, because inhibition of succinate dehydrogenase with nitropropionic acid did not block it. Of the [3-13C]lactate that reached the brain, 20% underwent this reaction, which probably involved neuronal mitochondrial malic enzyme. The activities of mitochondrial malic enzyme, fumarase, and lactate dehydrogenase were high enough to account for the formation of [2-13C]lactate in neurons. Neuronal pyruvate carboxylation was confirmed by the higher specific activity of glutamate than of glutamine after intrastriatal injection of [1-14C]pyruvate into anesthetized mice. This procedure also demonstrated equilibration of malate, formed through pyruvate carboxylation, with fumarate. The demonstration of neuronal pyruvate carboxylation demands reconsideration of the metabolic interrelationship between neurons and glia.
113 citations
TL;DR: It is shown that cultured cerebellar granule neurons form releasable [14C]glutamate from H14CO3− and [1- 14C]pyruvate via pyruVate carboxylation, probably mediated by malic enzyme, and thus entails a revision of the current view of glial–neuronal interactions and the importance of the glutamine cycle.
Abstract: Release of transmitter glutamate implies a drain of alpha-ketoglutarate from neurons, because glutamate, which is formed from alpha-ketoglutarate, is taken up by astrocytes. It is generally believed that this drain is compensated by uptake of glutamine from astrocytes, because neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates, which requires pyruvate carboxylation. Here we show that cultured cerebellar granule neurons form releasable [(14)C]glutamate from H(14)CO(3)(-) and [1-(14)C]pyruvate via pyruvate carboxylation, probably mediated by malic enzyme. The activity of pyruvate carboxylation was calculated to be approximately one-third of the pyruvate dehydrogenase activity in neurons. Furthermore, intrastriatal injection of NaH(14)CO(3) or [1-(14)C]pyruvate labeled glutamate better than glutamine, showing that pyruvate carboxylation occurs in neurons in vivo. This means that neurons themselves to a large extent may support their release of glutamate, and thus entails a revision of the current view of glial-neuronal interactions and the importance of the glutamine cycle.
106 citations
TL;DR: This work has evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenases (E1) component by PDK2 and PDK3.
105 citations
TL;DR: In this article, it was shown that the [4Fe-4S]-1+ cluster of PFL-AE is the catalytically relevant cluster, and that this cluster provides the electron necessary for AdoMetdependent glycyl radical generation.
Abstract: Pyruvate formate-lyase activating enzyme (PFL-AE), which generates the catalytically essential glycyl radical on PFL (Scheme 1),1 is a representative member of an emerging group of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) as required cofactors in radical generation. This group includes related activating enzymes such as the anaerobic ribonucleotide reductase activating enzyme (aRNR-AE) from E. coli,2 as well as biotin synthase,3,4 lipoic acid synthase,5,6 and lysine aminomutase (LAM).7 Though diverse in function, these enzymes have been proposed to have in common key mechanistic features including the generation of an intermediate 5′-deoxyadenosyl radical that initiates catalysis by hydrogen atom abstraction. Isotopic labeling has provided indirect evidence for such a mechanism for PFL-AE and LAM.8,9 Recently, elegant work by Frey and co-workers has provided direct spectroscopic evidence for an allylic analogue of the 5′-deoxyadenosyl radical for LAM.10 A central question surrounding this group of enzymes is the mechanism by which the iron-sulfur clusters participate in generation of the 5′-deoxyadenosyl radical intermediate. A variety of iron-sulfur clusters, including [2Fe-2S], [3Fe-4S], and [4Fe-4S], have been identified in these AdoMet-dependent enzymes.2-7,11-13 It has been difficult, however, to identify unequivocally the catalytically relevant cluster. A [4Fe-4S]1+ has been implicated as the active cluster for aRNR,2a and LAM containing a [4Fe-4S]1+ EPR signal has been shown to be catalytically active.7a We report here that for PFL-AE under conditions of limiting reductant, each [4Fe-4S]1+ cluster is capable of generating a single glycyl radical on PFL. Our results provide the first direct quantitative spectroscopic evidence that the [4Fe-4S]1+ of PFL-AE is the catalytically relevant cluster, and that this cluster provides the electron necessary for AdoMetdependent glycyl radical generation. PFL-AE isolated under anaerobic conditions contains primarily [3Fe-4S]+ clusters, as identified by UV-vis, EPR, and resonance Raman spectroscopies.13 The [3Fe-4S]+ cluster accounts for ∼62% of the total iron, and is characterized by an axial EPR signal centered at g ) 2.02.13 Upon reduction with dithionite, EPR spectra indicate that <20% of the reduced [4Fe-4S]1+ is generated, with the remainder of the clusters being in an EPR-silent state with UV-vis spectral properties characteristic of [4Fe-4S]2+ clusters.14 We have now found that illumination in the presence of 5-deazariboflavin allows nearly quantitative reduction to the [4Fe-4S]1+ state in a time-dependent manner, as indicated by spin quantitation of the resulting EPR signals (Figure 1A).15 As indicated by Scheme 1, single turnover conditions for PFL-AE can be achieved by limiting the amount of reductant. The clean conversion to [4Fe-4S]1+ provided by photoreduction has allowed us to carry out single turnover experiments for glycyl radical production, since removing illumination eliminates the exogenous reductant. PFL-AE was photoreduced for various times, after which a 10-fold excess of AdoMet was added and the sample was wrapped in aluminum foil to prevent further reduction.15 Each sample was then split into two halves and equimolar PFL was added to one-half in the dark. EPR spectra were recorded to detect formation of [4Fe-4S]1+ and glycyl radical in these samples. Figure 1A shows, from bottom to top, the 12 K EPR spectra of PFL-AE photoreduced in the presence of 5-deazariboflavin for 0, 1, 2, 5, 10, and 30 min.16 Quantitation of these EPR signals results in 0, 2.8((0.5), 17((2), 28((3), 41((4), and 54((5) μM spins, respectively.17 The nearly
100 citations
TL;DR: It is concluded that targeted regulation of PDK4 isoenzyme expression in skeletal muscle in response to starvation and refeeding underlies the modulation of the regulatory characteristics ofPDK in vivo.
Abstract: Using immunoblot analysis with antibodies raised against recombinant pyruvate dehydrogenase kinase (PDK) isoenzymes PDK2 and PDK4, we demonstrate selective changes in PDK isoenzyme expression in slow-twitch versus fast-twitch skeletal muscle types in response to prolonged (48 h) starvation and refeeding after starvation. Starvation increased PDK activity in both slow-twitch (soleus) and fast-twitch (anterior tibialis) skeletal muscle and was associated with loss of sensitivity of PDK to inhibition by pyruvate, with a greater effect in anterior tibialis. Starvation significantly increased PDK4 protein expression in both soleus and anterior tibialis, with a greater response in anterior tibialis. Starvation did not effect PDK2 protein expression in soleus, but modestly increased PDK2 expression in anterior tibialis. Refeeding for 4 h partially reversed the effect of 48-h starvation on PDK activity and PDK4 expression in both soleus and anterior tibialis, but the response was more marked in soleus than in anterior tibialis. Pyruvate sensitivity of PDK activity was also partially restored by refeeding, again with the greater response in soleus. It is concluded that targeted regulation of PDK4 isoenzyme expression in skeletal muscle in response to starvation and refeeding underlies the modulation of the regulatory characteristics of PDK in vivo. We propose that switching from a pyruvate-sensitive to a pyruvate-insensitive PDK isoenzyme in starvation (a) maintains a sufficiently high pyruvate concentration to ensure that the glucose-->alanine-->glucose cycle is not impaired, and (b) may 'spare' pyruvate for anaplerotic entry into the tricarboxylic acid cycle to support the entry of acetyl-CoA derived from fatty acid (FA) oxidation into the tricarboxylic acid cycle. We further speculate that FA oxidation by skeletal muscle is both forced and facilitated by upregulation of PDK4, which is perceived as an essential component of the operation of the glucose-FA cycle in starvation.
95 citations
TL;DR: Recombinant S. cerevisiae strains, with elevated levels of the enzymes of lower glycolysis (glyceraldehyde‐3‐phosphate dehydrogenase, phosphoglycerate mutase, phosphate kinase, enolase, pyruvate kinase) were physiologically characterized and the physiological behaviour of the recombinant strains and the host strain was similar.
Abstract: Recombinant S. cerevisiae strains, with elevated levels of the enzymes of lower glycolysis (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, phosphoglycerate kinase, enolase, pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase) were physiologically characterized. During growth on glucose the enzyme levels in the recombinant strains (YHM4 and YHM7) were 1.1–3.4-fold higher than in the host strain (CEN.PK.K45). The recombinant strains were grown in aerobic or anaerobic batch cultures on glucose or a mixture of glucose and galactose. The specific ethanol production rates in the recombinant strains were the same as for the host strain and the physiological behaviour of the recombinant strains and the host strain was similar. When the cellular demand for ATP was increased by means of glucose pulses (final concentrations of 3.9 g/l or 2.0 g/l, respectively) to aerobic chemostat cultures maintained at a dilution rate of 0.08/h, the specific carbon dioxide production rate (qCO2) of CEN.PK.K45 accelerated at 6×10−3 mmol/g/min2 during the first 15 min, whereas during the same time period the qCO2 of YHM7 accelerated twice as fast at 12×10−3 mmol/g/min2, indicating a higher fermentative capacity in the recombinant strain. Copyright © 2000 John Wiley & Sons, Ltd.
85 citations
TL;DR: PDHK isoform PDHK1 is identified as being of more potential regulatory importance for glucose oxidation in the adult compared with the neonatal heart, and cardiac PDHK4 as a PDHK isoforms whose expression is specifically responsive to changes in lipid supply, suggesting that its up-regulation during early post-natal life may be the perinatal switch to use fatty acids as the energy source.
Abstract: Activation of the pyruvate dehydrogenase (PDH) complex (PDHC) promotes glucose disposal, whereas inactivation conserves glucose. The PDH kinases (PDHKs) regulate glucose oxidation through inhibitory phosphorylation of PDHC. The adult rat heart contains three PDHK isoforms PDHK1, PDHK2 and PDHK4. Using Western-blot analysis, with specific antibodies raised against individual recombinant PDHK1, PDHK2 and PDHK4, the present study investigated PDHK isoform expression in the developing rat heart and adulthood. We identified clear differences in the patterns of protein expression of each of these PDHK isoforms during the first 3 weeks of post-natal development, with most marked up-regulation of isoforms PDHK1 and PDHK4. Distinctions between the three cardiac PDHK isoforms were also demonstrated with respect to post-neonatal maturational up-regulation; with greatest up-regulation of PDHK1 and least up-regulation of PDHK4 from the post-neonatal period until maturity. The study also examined the role of thyroid hormone status and lipid supply on PDHK isoform expression. We observed marked selective increases in the amount of PDHK4 protein present relative to total cardiac protein in both hyperthyroidism and high-fat feeding. Overall, our data identify PDHK isoform PDHK1 as being of more potential regulatory importance for glucose oxidation in the adult compared with the neonatal heart, and cardiac PDHK4 as a PDHK isoform whose expression is specifically responsive to changes in lipid supply, suggesting that its up-regulation during early post-natal life may be the perinatal switch to use fatty acids as the energy source. We also identify regulation of pyruvate sensitivity of cardiac PDHK as a physiological variable, a change in which requires factors in addition to a change in lipid supply.
TL;DR: Methyl substitution of the piperazine at the 2- and 5-positions markedly increased the potency of the lead compound (>1,000-fold) and oral bioavailability of the compounds in this series is good and is optimal when the 4-position of thepiperazine is substituted with an electron-poor benzoyl moiety.
Abstract: N‘-Methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC50 = 180 μM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S,R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibi...
TL;DR: Comparison of the enzymatic characteristics of the heterologously expressed human and rat dehydrogenases with those of purified rat liver trimethylaminobutyraldehyde dehydrogenase revealed that the three enzymes have highly similar substrate specificities.
TL;DR: Small electron-withdrawing groups on the ortho position of the anilide increased potency 20-40-fold and could be of utility to ameliorate conditions of inappropriate blood lactate elevation.
Abstract: The optimization of a series of anilide derivatives of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase (PDHK) is described that started from N-phenyl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 1 (IC50 = 35 ± 1.4 μM). It was found that small electron-withdrawing groups on the ortho position of the anilide, i.e., chloro, acetyl, or bromo, increased potency 20−40-fold. The oral bioavailability of the compounds in this series is optimal (as measured by AUC) when the anilide is substituted at the 4-position with an electron-withdrawing group (i.e., carboxyl, carboxyamide, and sulfoxyamide). N-(2-Chloro-4-isobutylsulfamoylphenyl)-(R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (10a) inhibits PDHK in the primary enzymatic assay with an IC50 of 13 ± 1.5 nM, enhances the oxidation of [14C]lactate into 14CO2 in human fibroblasts, lowers blood lactate levels significantly 2.5 and 5 h after oral doses as low as 30 μmol/kg, and increases the ex vivo activity of...
TL;DR: To elucidate the mechanism involved, site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer is performed and it is postulated that His-121 is one residue involved in kinase function.
Abstract: Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.
TL;DR: The results indicate that no other monocarboxylate permease is able to efficiently transport pyruvate in S. cerevisiae and that the mutant with a yeast genomic library allowed the isolation of the gene JEN1 (YKL217w), which restored pyruVate uptake.
Abstract: Pyruvate uptake in Saccharomyces cerevisiae was not observed at 0 degrees C and was prevented by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The initial uptake rate of S. cerevisiae kyokai No. 901 was maximum at pH 6 and Km = 4.1 mM. It seemed that lactate inhibited the pyruvate uptake competitively from the results of the Lineweaver-Burk plots. The inhibition constant (Ki) in the presence of 3 mM lactate was 1.6 mM. The pyruvate uptake was inhibited by D-glucose and deoxyglucose, but not by L-glucose, acetate or ethanol. Mutants of laboratory strain No. 5022 ((a) his(2,6), ura3) deficient in pyruvate uptake were isolated from fluoropyruvate resistant mutants. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 (YKL217w), which restored pyruvate uptake. Disruption of JEN1 abolished the uptake of pyruvate and gained the resistance against fluoropyruvate. The results indicate that no other monocarboxylate permease is able to efficiently transport pyruvate in S. cerevisiae.
TL;DR: Alanine dehydrogenase seems to be induced during growth with taurine, and was purified about 24-fold to apparent homogeneity in a three-step purification, suggesting a homo-hexameric structure.
Abstract: Alanine dehydrogenase [l-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of l-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0–11.5 for oxidative deamination of alanine. The apparent K
m values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.
TL;DR: In isotopic experiments, the labeling pattern of glutamate opens a window on hepatic metabolism, particularly the citric acid cycle, gluconeogenesis and fatty acid oxidation, and may allow the identification of patients who might benefit from the intraperitoneal administration of insulin, or from recently developed antidiabetic drugs.
Abstract: In isotopic experiments, the labeling pattern of glutamate opens a window on hepatic metabolism, particularly the citric acid cycle, gluconeogenesis and fatty acid oxidation. This is because glutamate is in isotopic equilibrium with alpha-ketoglutarate, whose labeling pattern is influenced by the following: 1) the contributions of glucose and fatty acids to acetyl-CoA, 2) the relative contributions of pyruvate carboxylase and pyruvate dehydrogenase to the entry of pyruvate carbon into the citric acid cycle, and 3) the rate of gluconeogenesis in relation to citric acid cycle activity. In humans and primates, hepatic glutamate can be sampled noninvasively via urinary phenylacetylglutamine, which is formed in liver from phenylacetate (a side product of phenylalanine catabolism) and glutamine (which equilibrates with liver glutamate and alpha-ketoglutarate). The (14)C- or (13)C-labeling pattern of the glutamate moiety of phenylacetylglutamine can be measured by sequential degradations to (14)CO(2), gas chromatography-mass spectrometry or nuclear magnetic resonance (NMR). When phenylacetylglutamine is labeled from singly labeled [(14)C]- or [(13)C]substrates, relative metabolic rates can be computed from the labeling pattern using Landau's model. In diabetic patients infused with [3-(13)C]pyruvate, the noninvasive sampling of hepatic glutamate via phenylacetylglutamine allows one to test the degree of liver insulinization via the (pyruvate carboxylase)/(pyruvate dehydrogenase) activity ratio. This ratio regulates gluconeogenesis in part. Its measurement may allow the identification of patients who might benefit from the intraperitoneal administration of insulin, or from recently developed antidiabetic drugs.
TL;DR: Regulation of currently identified genes involved in pyruvate metabolism of Kluyveromyces lactis strain CBS 2359 was studied and the activity of the K. lactis pyruVate dehydrogenase complex appeared to be regulated at the level of KlPDA1 transcription, suggesting that regulation is mostly achieved at the transcriptional level.
Abstract: Regulation of currently identified genes involved in pyruvate metabolism of Kluyveromyces lactis strain CBS 2359 was studied in glucose-limited, ethanol-limited and acetate-limited chemostat cultures and during a glucose pulse added to a glucose-limited steady-state culture. Enzyme activity levels of the pyruvate dehydrogenase complex, pyruvate decarboxylase, alcohol dehydrogenase, acetyl-CoA synthetase and glucose-6-phosphate dehydrogenase were determined in all steady-state cultures. In addition, the mRNA levels of KlADH1-4, KlACS1, KlACS2, KlPDA1, KlPDC1 and RAG1 were monitored under steady-state conditions and during glucose pulses. In K. lactis, as in Saccharomyces cerevisiae, enzymes involved in glucose utilization (glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, pyruvate decarboxylase) showed the highest expression levels on glucose, whereas enzymes required for ethanol or acetate consumption (alcohol dehydrogenase, acetyl-CoA synthetase) showed the highest enzyme activities on ethanol. In cases where mRNA levels were determined, these corresponded well with the corresponding enzyme activities, suggesting that regulation is mostly achieved at the transcriptional level. Surprisingly, the activity of the K. lactis pyruvate dehydrogenase complex appeared to be regulated at the level of KlPDA1 transcription. The conclusions from the steady-state cultures were corroborated by glucose pulse experiments. Overall, expression of the enzymes of pyruvate metabolism in the Crabtree-negative yeast K. lactis appeared to be regulated in the same way as in Crabtree-positive S. cerevisiae, with one notable exception: the PDA1 gene encoding the E1alpha subunit of the pyruvate dehydrogenase complex is expressed constitutively in S. cerevisiae.
TL;DR: Overall, these data demonstrate that small molecules acting at diverse sites can inhibit PDK activity and are shown to be directly inhibited by a novel mechanism; the addition of supplemental recombinant PDKs.
TL;DR: A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.
Abstract: Cytosolic pyruvate kinase (PK(c)) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7 micromol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240 kDa homotetramer composed of subunits of 57 kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of V(max)/K(m) for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH 6.9 and 7.5. PK(c) activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg(2+) and K(+) respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg(2+) and K(+) (K(m) values of 0.098, 0.12, 0.27 and 0.91 mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PK(c) by L-glutamate. The allosteric features of banana PK(c) are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807-816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.
TL;DR: The results indicate that BCKD kinase, similar to pyruvate dehydrogenase kinase isozymes, belongs to the superfamily of ATPase/kinase.
TL;DR: Comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized and are being tested by combined biochemical, genetic and molecular biological experiments, providing insights into how acetyl -CoA metabolism is regulated.
Abstract: We have characterized the expression of potential acetyl-CoA-generating genes (acetyl-CoA synthetase, pyruvate decarboxylase, acetaldehyde dehydrogenase, plastidic pyruvate dehydrogenase complex and ATP-citrate lyase), and compared these with the expression of acetyl-CoA-metabolizing genes (heteromeric and homomeric acetyl-CoA carboxylase). These comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. These hypotheses are being tested by combined biochemical, genetic and molecular biological experiments, which is providing insights into how acetyl-CoA metabolism is regulated.
TL;DR: The ability of glutathione to block inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by scavenging electrophilic intermediates, generated by the mitochondrial membrane-catalyzed oxidation of DHBT-NE-1, forming glutathionyl conjugates are discussed.
Abstract: The major initial product of the oxidation of norepinephrine (NE) in the presence of L-cysteine is 5-S-cysteinylnorepinephrine which is then further easily oxidized to the dihydrobenzothiazine (DHBT) 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1, 4-benzothiazine-3-carboxylic acid (DHBT-NE-1). When incubated with intact rat brain mitochondria, DHBT-NE-1 evokes rapid inhibition of complex I respiration without affecting complex II respiration. DHBT-NE-1 also evokes time- and concentration-dependent irreversible inhibition of NADH-coenzyme Q(1) (CoQ(1)) reductase, the pyruvate dehydrogenase complex (PDHC), and alpha-ketoglutarate dehydrogenase (alpha-KGDH) when incubated with frozen and thawed rat brain mitochondria (mitochondrial membranes). The time dependence of the inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by DHBT-NE-1 appears to be related to its oxidation, catalyzed by an unknown component of the inner mitochondrial membrane, to electrophilic intermediates which bind covalently to active site cysteinyl residues of these enzyme complexes. The latter conclusion is based on the ability of glutathione to block inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by scavenging electrophilic intermediates, generated by the mitochondrial membrane-catalyzed oxidation of DHBT-NE-1, forming glutathionyl conjugates, several of which have been isolated and spectroscopically identified. The possible implications of these results to the degeneration of neuromelanin-pigmented noradrenergic neurons in the locus ceruleus in Parkinson's disease are discussed.
TL;DR: Enzymatic activities and energetic states in fibroblasts from lipoamide dehydrogenase-deficient patients representing three different phenotypes and genotypes were evaluated, and direct relationships between clinical parameters such as age of onset and disease severity and biochemical characteristics were identified.
TL;DR: The acute toxicity of CCl3NO2 in mice is due to the parent compound or metabolites other than CHCl2NO2 or CH2ClNO2 and may be associated with inhibition of the pyruvate dehydrogenase complex and elevated oxyhemoglobin.
Abstract: Chloropicrin (CCl3NO2) is a widely used soil fumigant with an unknown mechanism of acute toxicity. We investigated the possible involvement of dechlorination in CCl3NO2 toxicity by considering its metabolism, inhibition of pyruvate and succinate dehydrogenases, cytotoxicity in cultured cells, and interaction with hemoproteins. In a newly discovered pathway, CCl3NO2 is metabolized to thiophosgene, which is characterized as the cyclic cysteine adduct (raphanusamic acid) in the urine of mice. CCl3NO2 inhibits porcine heart pyruvate dehydrogenase complex (IC-50 4 μM) and mouse liver succinate dehydrogenase complex (IC-50 13 μM), whereas its dehalogenated metabolites (CHCl2NO2 and CH2ClNO2 are more than 10 times less effective. The inhibitory potency of CCl3NO2 for these dehydrogenase complexes is similar to that of captan, folpet, and dichlone fungicides (IC-50 2–6 μM). CCl3NO2 cytotoxity with Hepa 1c1c7 mouse hepatoma cells (IC-50 9 μM) is not correlated with glutathione depletion. Mice treated intraperitoneally with CCl3NO2 at 50 mg/kg but not with an equivalent dose of CHCl2NO2 show increased concentrations of oxyhemoglobin in liver. The acute toxicity of CCl3NO2 in mice is due to the parent compound or metabolites other than CHCl2NO2 or CH2ClNO2 and may be associated with inhibition of the pyruvate dehydrogenase complex and elevated oxyhemoglobin. © 1999 John Wiley & Sons, Inc. J Biochem Toxicol 14: 26–32, 2000
TL;DR: Pancreatic islet beta-cells are poised to generate metabolic messengers in the mitochondria that link glucose metabolism to insulin exocytosis through the tight coupling of glycolysis to mitochondrial activation, suggesting that NADH availability restricts the conversion of pyruvate to lactate in the beta cell.
Abstract: Pancreatic islet beta-cells are poised to generate metabolic messengers in the mitochondria that link glucose metabolism to insulin exocytosis. This is accomplished through the tight coupling of glycolysis to mitochondrial activation. The messenger molecules ATP and glutamate are produced after the metabolism of glycolysis-derived pyruvate in the mitochondria. The entry of monocarboxylates such as pyruvate into the beta cell is limited, explaining why overexpression of monocarboxylate transporters unravels pyruvate-stimulated insulin secretion. NADH generated by glycolysis is efficiently reoxidized by highly active mitochondrial shuttles rather than by lactate dehydrogenase. Overexpression of this enzyme does not alter glucose-stimulated insulin secretion, suggesting that NADH availability restricts the conversion of pyruvate to lactate in the beta cell. These metabolic features permit the fuel function of glucose to be extended to the generation of signaling molecules, which increases cytosolic Ca2+ and promotes insulin exocytosis.
TL;DR: Results provide the first evidence suggesting that there is the regional difference in the abundance of mRNAs for PDK isoenzymes in rat brain and that the levels of m RNAs for the isoenZymes were affected by aging.
TL;DR: It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other, which has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.
Abstract: The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (alpha2beta2) of E1alpha and E1beta polypeptide chains. The domain structure of the E1alpha and E1beta chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha2beta2). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.