Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase

It has been proposed that "Glu238" within the N-box of pyruvate dehydrogenase kinase (PDK) is a base catalyst. The pH dependence of k(cat) of Arabidopsis thaliana PDK indicates that ionizable groups with pK values of 6.2 and 8.4 are necessary for catalysis, and the temperature dependence of these values suggests that the acidic pK is due to a carboxyl- or imidazole-group. The E238 and K241 mutants had elevated K(m,ATP) values. The acidic pK value of the E238A mutant was shifted to 5.5. The H233A, L234H, and L234A mutants had the same pK values as wild-type AtPDK, contrary to the previous proposal of a "Glu-polarizing" His. Instead, we suggest that the conserved Glu, Lys, and Asn residues of the N-box contribute to coordinating Mg2+ in a position critical for formation of the PDK-MgATP-substrate ternary complex.     

Expression of human pyruvate carboxylase in insect cells using the baculovirus system

Constructs containing cDNA encoding human pyruvate carboxylase (PC) with and without a hexahistidine (6x His) tag at the N-terminal of the mature enzyme have been cloned under the control of the polyhedrin promoter. These two constructs were co-transfected with the baculovirus genome into Sf9 cells to produce recombinant baculoviruses harbouring human PC cDNA. The expression of human PC under the control of the polyhedrin promoter was found to be at its highest level at 4 days post-infection. The expressed material accounted for up to 70% of total cellular protein with 5% of this expressed material being found in the soluble fraction. The recombinant human 6x His-PC isolated with a purity of approximately 50% using a Ni-NTA agarose column was found to have the specific activity of 7U/mg, which was similar to that produced from a 293T stable line [Biochem. Biophys. Res. Commun. 266 (1999) 512]. This is the first report of a heterologous expression system for recombinant human PC.     

Identification of pyruvate dehydrogenase kinase 1 inhibitors with anti-osteosarcoma activity

Overexpression of pyruvate dehydrogenase kinases (PDKs), especially PDK1 has been observed in a variety of cancers. Thus, targeting PDK1 offers an attractive opportunity for the development of cancer therapies. In this letter, we reported the identification of two novel PDK1 inhibitors as anti-osteosarcoma agents. We found that TM-1 and TM-2 inhibited PDK1 with the IC₅₀ values of 2.97 and 3.41?μM, respectively. Furthermore, TM-1 and TM-2 dose-dependently reduced phosphorylation of pyruvate dehydrogenase complex in MG-63 osteosarcoma cells. Finally, TM-1 and TM-2 were found to inhibit the proliferation of MG-63 cells with the EC₅₀ values of 14.5, and 11.0?μM, respectively, meaning TM-1 and TM-2 could be promising leads for the discovery of potent PDK1 inhibitors.     

Dichloroacetophenones targeting at pyruvate dehydrogenase kinase 1 with improved selectivity and antiproliferative activity: Synthesis and structure-activity relationships

Dichloroacetophenone is a pyruvate dehydrogenase kinase 1 (PDK1) inhibitor with suboptimal kinase selectivity. Herein, we report the synthesis and biological evaluation of a series of novel dichloroacetophenones. Structure-activity relationship analyses (SARs) enabled us to identify three potent compounds, namely 54, 55, and 64, which inhibited PDK1 function, activated pyruvate dehydrogenase complex, and reduced the proliferation of NCI-H1975 cells. Mitochondrial bioenergetics assay suggested that 54, 55, and 64 enhanced the oxidative phosphorylation in cancer cells, which might contribute to the observed anti-proliferation effects. Collectively, these results suggested that 54, 55, and 64 could be promising compounds for the development of potent PDK1 inhibitors.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

Postischemic inhibition of cerebral cortex pyruvate dehydrogenase

Postischemic, mitochondrial respiratory impairment can contribute to prolonged intracellular lactic acidosis, secondary tissue deenergization, and neuronal cell death. Specifically, reperfusion-dependent inhibition of pyruvate dehydrogenase (PDH) may determine the degree to which glucose is metabolized aerobically vs. anaerobically. In this study, the maximal activities of pyruvate and lactate dehydrogenase (LDH) from homogenates of canine frontal cortex were measured following 10 min of cardiac arrest and systemic reperfusion from 30 min to 24 h. Although no change in PDH activity occurred following ischemia alone, a 72% reduction in activity was observed following only 30 min of reperfusion and a 65% inhibition persisted following 24 h of reperfusion. In contrast, no significant alteration in LDH activity was observed in any experimental group relative to nonarrested control animals. A trend toward reversal of PDH inhibition was observed in tissue from animals treated following ischemia with acetyl-L-carnitine, a drug previously reported to inhibit brain protein oxidation, and lower postischemic cortical lactate levels and improve neurological outcome. In vitro experiments indicate that PDH is more sensitive than LDH to enzyme inactivation by oxygen dependent free radical-mediated protein oxidation. This form of inhibition is potentiated by either elevated Ca²⁺ concentrations or substrate/cofactor depletion. These results suggest that site-specific protein oxidation may be involved in reperfusion-dependent inhibition of brain PDH activity.     

Regulation of Phosphate Incorporation into Four Brain Phosphoproteins that Are Affected by Experience

Various regulators of protein kinase activities were tested for their effects on the in vitro transfer of phosphate from [gamma-32P]ATP to four proteins of rat brain synaptic particulate preparations. One protein, of apparent molecular weight 44,000, accepted 32P in the presence of 8 mM EDTA and no added Mg2+. It was the major phosphoprotein of brain mitochondria. Its phosphorylation was inhibited by pyruvate and stimulated by K+, and it comigrated in electrophoretic gels with authentic alpha-subunit of pyruvate: lipoamide oxidoreductase (decarboxylating) (EC 1.2.4.1) from bovine heart. The major kinase acting on three proteins of apparent molecular weights 24,000, 21,000, and 19,000 was stimulated by Ca2+, by preincubation with phospholipase C, and by 12-tetradecanoyl 4-beta-phorbol 13-acetate. Phosphorylation of these lower-molecular-weight proteins was inhibited by ACTH1-24, by cyclic 3',5'-adenosine monophosphate, and by 50 microM trifluoperazine. The stimulatory effect of Ca2+ was antagonized by calmodulin. The kinase in question appears to be B-50 protein kinase or protein kinase C.     

In vitro cytotoxicity screening to identify novel anti-osteosarcoma therapeutics targeting pyruvate dehydrogenase kinase 2

Pyruvate dehydrogenase kinases (PDKs) act as negative modulator of mitochondrial pyruvate dehydrogenase complex (PDC) and play a crucial role in the regulation of oxidative glycolysis, which recently have been considered as a potential drug target for varying types of cancer and diabetes. Herein, we describe the discovery and biological validation of novel anti-osteosarcoma therapeutics targeting PDK2. We identified 14 anti-osteosarcoma compounds from an in-house small molecule library, which were then evaluated in a PDK2 kinase inhibition assay. We found that compounds with 2-((4-oxo-6-((4-phenylpiperazin-1-yl)methyl)-4H-pyran-3-yl)oxy)acetamide moiety showed promising inhibitory potencies to PDK2. Especial for 12, which bound to PDK2 with a K_d value of 2.3 µM, and inhibited PDK2 activity with an EC₅₀ value of 1.1 µM. In addition, 12 selectively inhibited PDK2, the selectivity indexes are 10.6, 22.0, and 60.9 for PDK2 as compared to PDK1, 2 and 4, respectively. The MTT assay suggested that 12 reduced MG-63 cancer cell proliferation with an IC₅₀ value of 4.7 µM. All these observations indicated that 12 was a novel anti-osteosarcoma therapeutic, which deserved for further investigation.     

Assay of the pyruvate dehydrogenase complex by coupling with recombinant chicken liver arylamine N-acetyltransferase

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.     

Discovery of novel pyruvate dehydrogenase kinases inhibitors by screening of an in-house small molecule library for anti-lung cancer therapeutics

Pyruvate dehydrogenase kinases (PDKs) are widely over-expressed in various human solid cancers, making them attractive therapeutic targets for cancer treatment. Herein, we report the identification of structurally novel PDKs inhibitors by screening of an in-house small molecule library. Biochemical assay indicated that the identified compounds 1–4 inhibited PDK1 activity with EC₅₀ values of 0.50, 1.99, 4.64, and 0.42?µM, respectively. The ITC analysis suggested that the identified compounds 1–4 were pan-isoform PDK inhibitors, which bound to and inhibited the four PDK isoforms. Moreover, 1–4 dose-dependently reduced pyruvate dehydrogenase complex phosphorylation in NCI-H1975 cell. Molecular docking suggested that the most potent compound 4 docked well in the ATP binding pocket of the four PDK isoforms, forming direct hydrogen bond interactions with the conserved amino acids Thr and Asp in ATP binding pocket of PDKs. The cell viability assay demonstrated that 4 potently blocked NCI-H1975 cell proliferation (IC₅₀?=?3.32?µM), but had little effect on human normal lung cell MRC-5 even with the tested concentration up to 40?µM. All the data demonstrated that 4 was a promising lead for the development of structurally novel PDKs inhibitor for the cancer treatment.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Monitoring phosphorylation of the pyruvate dehydrogenase complex

The pyruvate dehydrogenase multienzyme complex (PDC) is a key regulatory point in cellular metabolism linking glycolysis to the citric acid cycle and lipogenesis. Reversible phosphorylation of the pyruvate dehydrogenase enzyme is a critical regulatory mechanism and an important point for monitoring metabolic activity. To directly determine the regulation of the PDC by phosphorylation, we developed a complete set of phospho-antibodies against the three known phosphorylation sites on the E1 alpha subunit of pyruvate dehydrogenase (PDHE1α). We demonstrate phospho-site specificity of each antibody in a variety of cultured cells and tissue extracts. In addition, we show sensitivity of these antibodies to PDH activity using the pyruvate dehydrogenase kinase-specific inhibitor dichloroacetate. We go on to use these antibodies to assess PDH phosphorylation in a patient suffering from Leigh’s syndrome. Finally, we observe changes in individual phosphorylation states following a small molecule screen, demonstrating that these reagents should be useful for monitoring phosphorylation of PDHE1α and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli.     

Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus

The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate in mitochondria. The PDC activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by specific kinases (PDK) and phosphatases (PDP). In this study, the regulatory mechanisms of PDC were examined in skeletal muscle of the spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rat before and after the onset of diabetes. The Long-Evans Tokushima Otsuka (LETO) rat was used as control. Plasma glucose and insulin concentrations were at normal levels in both groups at 8 weeks of age but were significantly higher in OLETF than in LETO rats at 25 weeks of age (1.2-fold for glucose and 15-fold for insulin), indicating development of diabetes in the former. Plasma free fatty acids were 1.6-fold concentrated and the skeletal muscle PDC activity state was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidation in OLETF rats even before the onset of diabetes. The PDK activity and the abundance of the PDK isoform 4 protein as well as mRNA were greater in OLETF rats at both ages. Conversely, the abundance of the PDP isoform 1 protein and mRNA was less in OLETF than in LETO rats at both ages. These results suggest that concomitant greater PDK4 and less PDP1 expression in skeletal muscle of OLETF rats before the onset of diabetes are responsible for the lowering of the PDC activity and may be related with the development of diabetes mellitus.     

Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex

Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates. Enzyme turnover was measured by following NADH production spectrophotometrically or by a fluorescence assay on mitochondrial protein preparations in the range of 0.4 to 5.0 micro g per well. Activity is sensitive to known PDH inhibitors and remains regulated by phosphorylation and dephosphorylation after immunopurification because of the presence of bound PDH kinase(s) and phosphatase(s). It is shown that the immunocapture assay can be used to detect PDH deficiency in cell extracts of cultured fibroblasts from patients, making it useful in patient screens, as well as in the high-throughput format for discovery of new modulators of PDH functioning.     

Glutamate-Stimulated Phosphorylation of a Specific Protein in P2 Fractions of Rat Cerebral Cortex

Incubation of P2 fractions from rat cerebral cortex with 32Pi in the presence of L-glutamate caused an increased phosphorylation of a protein with apparent molecular weight of 43,000 (P43) as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. This glutamate-stimulated phosphorylation of P43 was already detectable 10 s after the addition of glutamate and was dependent on the concentrations of glutamate in the incubation medium. Other excitatory amino acids such as D-glutamate, L-aspartate, D,L-cysteic acid, L-cysteinesulfinic acid, and D,L-alpha-aminoadipic acid did not stimulate the phosphorylation of P43. In contrast, alpha-ketoglutarate and succinate stimulated the phosphorylation of this protein. Glutamate-stimulated phosphorylation of P43 seemed not to be mediated by either cAMP or cGMP and was inhibited by the presence of Ca2+ in the incubation medium. Experiments performed with metabolic inhibitors indicated that glutamate-stimulated protein phosphorylation is localized in mitochondria. This conclusion is supported by the occurrence of glutamate-stimulated phosphorylation of P43 in mitochondrial fractions from several peripheral tissues. The present results are consistent with the hypothesis that P43 is a component of the pyruvate dehydrogenase complex of mitochondria.     

Prediction of the binding site on E1 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The beta-subunit (E1beta) of the pyruvate decarboxylase (E1, alpha(2)beta(2)) component of the Bacillus stearothermophilus pyruvate dehydrogenase complex was comparatively modelled based on the crystal structures of the homologous 2-oxoisovalerate decarboxylase of Pseudomonas putida and Homo sapiens. Based on this homology modelling, alanine-scanning mutagenesis studies revealed that the negatively charged side chain of Glu285 and the hydrophobic side chain of Phe324 are of particular importance in the interaction with the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the complex. These results help to identify the site of interaction on the E1beta subunit and are consistent with thermodynamic evidence of a mixture of electrostatic and hydrophobic interactions being involved.     

Lysine-ketoglutarate reductase and saccharopine dehydrogenase from Arabidopsis thaliana: nucleotide sequence and characterization

We isolated the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, ED 1.5.1.9) from an Arabidopsis thaliana genomic DNA library based on the homology between the yeast biosynthetic genes encoding SDH (lysine-forming) or SDH (glutamate-forming) and Arabidopsis expressed sequence tags. A corresponding cDNA was isolated from total Arabidopsis RNA using RT-PCR and 5 and 3 Race. DNA sequencing revealed that the gene encodes a bifunctional protein with an amino domain homologous to SDH (lysine-forming), thus corresponding to LKR, and a carboxy domain homologous to SDH (glutamate-forming). Sequence comparison between the plant gene product and the yeast lysine-forming and glutamate-forming SDHs showed 25% and 37% sequence identity, respectively. No intracellular targeting sequence was found at the N-terminal or C-terminal of the protein. The gene is interrupted by 24 introns ranging in size from 68 to 352 bp and is present in Arabidopsis in a single copy. 5 sequence analysis revealed several conserved promoter sequence motifs, but did not reveal sequence homologies to either an Opaque 2 binding site or a Sph box. The 3-flanking region does not contain a polyadenylation signal resembling the consensus sequence AATAAA. The plant SDH was expressed in Escherichia coli and exhibited similar biochemical characteristics to those reported for the purified enzyme from maize. This is the first report of the molecular cloning of a plant LKR-SDH genomic and cDNA sequence.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.