Showing papers on "Oxoglutarate dehydrogenase complex published in 1990"
TL;DR: Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, establishing the enzyme to be a dimer of similar-sized protein chains.
Abstract: Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, with chromatography on DEAE-Sepharose, Blue Sepharose, and Mono-Q FPLC as principal steps. Final yield was 23% and purification about 13,000-fold, with a specific activity of 24,000 milliunits/mg. The subunit molecular weight is about 29,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the native protein molecular weight is about 54,000 as estimated by Sephadex G-100 chromatography, establishing the enzyme to be a dimer of similar-sized protein chains. The subunit N-terminal residue is methionine, and the alpha-amino group is free. The complete primary structure was determined by peptide analysis, based essentially on four different proteolytic treatments (Lys-specific protease, Glu-specific protease, Asp-specific protease, and CNBr). The protein chain is composed of 266 residues, with C-terminal glutamine. A microheterogeneity was detected at position 217, with both Cys and Tyr, in about equal amounts, from a preparation starting with a single placenta. No other subunit heterogeneities were detected. The protein is clearly but distantly related to insect alcohol dehydrogenases, characterized bacterial dehydrogenases of sugar metabolism, and bacterial and eukaryotic steroid dehydrogenases. Together, these results establish that placental 15-hydroxyprostaglandin dehydrogenase is a member of the short-chain nonmetalloenzyme alcohol dehydrogenase protein family. The protein has four cysteine residues (five with the positional microheterogeneity), but there is no evidence for functional importance of any of these residues.(ABSTRACT TRUNCATED AT 250 WORDS)
108 citations
TL;DR: In this article, the authors explored the role of 2-ketoglutarate dehydrogenase in the preferential and Ca2(+)-dependent stimulation of mitochondrial oxidative events in pancreatic islet cells.
106 citations
TL;DR: A substantial role for substrate dehydrogenation in the overall regulation of oxidative phosphorylation when substrates are available at near-physiological concentrations is interpreted to indicate a substantial role by Ca2+ of the enzymes ofosphorylation or of the respiratory chain in addition to the dehydrogenase activation.
Abstract: 1. We have examined systematically the relationship between the percentage reduction of cardiac mitochondrial NAD and the flux through oxidative phosphorylation, as measured by O2 uptake. Reduction of NAD was varied by varying the concentration of palmitoyl-L-carnitine, pyruvate, 2-oxoglutarate or glutamate in the presence of malate as the oxidizable substrate. 2. In the presence of ADP (State 3 respiration) there was a substantially linear positive relationship between O2 uptake and the percentage reduction of NAD. Coupled respiration in the absence of ADP also showed an increase with increasing NADH, with the exact shape of the relationship being variable. 3. When pyruvate and 2-oxoglutarate dehydrogenase activity were increased by increasing medium Ca2+ concentration within the range 5 nM to 1.23 microM, at non-saturating substrate concentrations, there was again a positive relationship between O2 uptake and the reduction of NAD; however, rates of O2 uptake tended to be higher at given values of NAD reduction when the incubation medium contained Ca2+. This is taken to indicate an activation by Ca2+ of the enzymes of phosphorylation or of the respiratory chain, in addition to the dehydrogenase activation. 4. When carboxyatractyloside plus ADP were used to generate 50% State 3 rates of O2 uptake with pyruvate or 2-oxoglutarate, sensitivity to Ca2+ was retained. However, when oligomycin plus 1 mM-ADP and 1 mM-ATP were used to generate 50% State 3, no such dependence was seen. 5. The results are interpreted to indicate a substantial role for substrate dehydrogenation in the overall regulation of oxidative phosphorylation when substrates are available at near-physiological concentrations.
94 citations
TL;DR: Branched-chain alpha-ketoacid dehydrogenase kinase was purified to homogeneity from rat liver and rat heart with high kinase activity by a modification of a method described previously.
82 citations
TL;DR: The molecular basis of one maple syrup urine disease family has been determined and the primary structure of the mature proteins deduced for the E1 alpha subunit of the human and rat liver branched-chain alpha-ketoacid dehydrogenase complex is identified.
72 citations
TL;DR: It is confirmed that primary biliary cirrhosis sera react with dihydrolipoamide succinyltransferase, the E2 component of α‐ketoglutarate dehydrogenase complex, and the data suggest that these autoantibodies are directed toward a functional domain of this enzyme.
65 citations
TL;DR: Inhibition of purified beef brain KDHC by VPA and its metabolites 2-n-propylpent-2-enoic acid and their coenzyme A (CoA) derivatives cause inactivation and inhibition of KDHC, which may explain the anticonvulsant and some toxic actions of VPA.
Abstract: The anticonvulsant valproic acid (VPA, 2-n-propylpentanoic acid) causes inhibition of the citric acid cycle and elevations of central nervous system (CNS) gamma-aminobutyric acid (GABA) levels, which correlates with anticonvulsant action. No unifying mechanism for these actions of VPA has won general acceptance. alpha-Ketoglutarate dehydrogenase complex (KDHC) is a critical control enzyme in the CNS. We hypothesized that VPA may be an inhibitor of this enzyme since decreased KDHC activity would reduce substrate flux through the citric acid cycle and may increase flux into GABA synthesis. To test this hypothesis, inhibition of purified beef brain KDHC by VPA and its metabolites 2-n-propylpent-2-enoic acid (delta 2,3 VPE) and their coenzyme A (CoA) derivatives were studied. Preincubation of the NADH-reduced enzyme with delta 2,3 VPE, VPA-CoA, and delta 2,3 VPE-CoA caused time-dependent inactivation, reversible by addition of CoA. Under steady-state conditions, delta 2,3 VPE and VPA-CoA were competitive inhibitors of KDHC and delta 2,3 VPE-CoA was a mixed inhibitor. These observations have implications for the molecular mechanisms of VPA action. VPA derivatives cause inactivation and inhibition of KDHC, which may explain the anticonvulsant and some toxic actions of VPA.
51 citations
50 citations
TL;DR: The native architectures of the pyruvate and 2-oxoglutarate dehydrogenase complexes have been investigated by cryoelectron microscopy of unstained, frozen-hydrated specimens and images do not appear consistent with a symmetric arrangement of the E1 and E3 subunits about the octahedrally symmetric E2 core.
49 citations
TL;DR: The finding that glutamate decarboxylase was essentially absent in six different strains of B. japonicum was consistent with the labelling results and indicated that conversion of glutamate to succinate via GAB is slow or nil.
Abstract: Summary: Previous studies with labelled N and C have indicated synthesis and accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions similar to those found in soybean nodules. Low 2-oxoglutarate dehydrogenase (OGDH) activity might have accounted for this observation, but similar levels of enzyme activity were found in bacteroids isolated anaerobically or aerobically and in cultured bacteria. However, OGDH from B. japonicum bacteroids was strongly inhibited by NADH, and the degree of inhibition depended on the NADH:NAD ratio. Determination of endogenous levels of NAD and NADH gave NADH:NAD ratios of 0.19 and 0.83 in bacteroids isolated under aerobic and anaerobic conditions, respectively. A ratio of 0.83 resulted in more than 50% inhibition of OGDH in vitro, and this would be consistent with channelling of 2-oxoglutarate to glutamate. [14C]Glutamate supplied to bacteroids was metabolized to CO2 slowly relative to the respiration of malate, and essentially no labelling of products of glutamate metabolism such as arginine, proline, glutamine and 4-aminobutyrate (GAB) was found. Attempts to trap 14C in GAB by supplying unlabelled GAB or transaminase inhibitors with [14C]glutamate were unsuccessful. The finding that glutamate decarboxylase was essentially absent in six different strains of B. japonicum was consistent with the labelling results and indicated that conversion of glutamate to succinate via GAB is slow or nil. The inhibition of OGDH by a high NADH:NAD ratio and the absence of the GAB shunt are complementary mechanisms which probably account for the accumulation of glutamate.
48 citations
TL;DR: Bovine kidney mitochondria were separated into matrix and membrane fractions by treatment with digitonin and Lubrol PX, and both multienzyme complexes must be associated with the inner membrane rather than located within the matrix space.
TL;DR: The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined and it is likely that the three genes of the oxoglUTarate dehydrogensase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogen enzyme complex in E. coli.
Abstract: The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. Previously the cloning in Escherichia coli of the gene encoding lipoamide dehydrogenase from A. vinelandii was reported [Westphal, A.H. & de Kok, A. (1988) Eur. J. Biochem. 172, 299-305]. The 3.2-kb fragment used for the sequence determination contained the main part of the gene encoding succinyltransferase. The complete E2o gene, as well as the gene encoding the 2-oxoglutarate dehydrogenase component, resided on a 14.7-kb fragment from which the 3.2-kb fragment was subcloned. The protein-coding sequence of the gene consists of 1200 bp (400 codons, including the AUG start codon and the UGA stop codon). It is separated from the gene encoding the 2-oxoglutarate dehydrogenase component by 42 bp. No E. coli-like promoter sequence was found. A putative ribosome-binding site is located 9-15 bp upstream from the start codon. No terminator sequences were found downstream of the stop codon. This makes it likely that the three genes of the oxoglutarate dehydrogenase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogenase complex in E. coli. The intact gene was subcloned from the 14.7-kb fragment and brought to high expression under the influence of the vector-encoded lacZ promoter. The similarity with the E. coli enzyme is high with 63% identity. Like the enzyme from E. coli, it consists of a single lipoyl-binding domain, a putative E1- and E3-binding domain and a catalytic domain. The main difference is found in a 31-residue sequence rich in alanine and proline located between the lipoyl domain and the putative E1- and E3-binding domain. This sequence, usually found in acetyltransferases and there identified as a highly mobile region by 1H-NMR, is replaced by a more polar, charged region in the E. coli enzyme.
TL;DR: An infant with moderate muscular hypotonia and congenital lactic acidosis died suddenly at the age of 3 months, and immunochemical analysis in skeletal muscle tissue and fibroblasts demonstrated a decrease in the amount of the α and β subunits of E1.
Abstract: An infant with moderate muscular hypotonia and congenital lactic acidosis died suddenly at the age of 3 months. Autopsy revealed no abnormalities responsible for this unexpected death. Measurement of mitochondrial enzymes involved in energy production indicated a severely decreased total pyruvate dehydrogenase complex (PDHC) activity in muscle tissue (0.23 nmoles · min−1 · mg protein−1, control range 2.8–8.7) and moderately decreased PDHC activity in fibroblasts (0.27 nmoles · min−1 · mg protein−1, control range 0.37–2.32). The activity of the first component E1 (pyruvate dehydrogenase) in muscle tissue was 10 times lower than that of controls (0.008 nmoles · min−1 · mg protein−1, control range 0.10–0.25). The activities of dihydrolipoyl dehydrogenase (E3) and various other mitochondrial enzymes were normal. Immunochemical analysis in skeletal muscle tissue and fibroblasts demonstrated a decrease in the amount of the α and β subunits of E1. The features of this patient are compared with those of other patients reported in the literature with immunochemically confirmed combined E1 α and β deficiency.
TL;DR: Thermoinducible expression was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E1p component, and the results support the view that His-602 is an active- site residue.
Abstract: The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.
01 Jan 1990
TL;DR: It is suggested that the interaction of the pyruvate dehydrogenase with acetylphosphinate involves the formation of a thiamin pyrophosphate-acetylph phosphinate adduct that resembles the normal reaction intermediate, 2-(1-carboxy-1-hydroxyethyl)thiamIn pyroph phosphate (alpha-lactylthiaminpyrophosphates)
Abstract: The pyruvate analogue acetylphosphinate (CH3-CO-PO2H2) inhibits the pyruvate dehydrogenase component (E1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex in a time-dependent process with biphasic reaction kinetics. The formation of an initial, rapidly reversible enzyme-inhibitor complex (EI) with an apparent Ki of 0.12 +/- 0.025 microM is followed by the conversion to a tighter complex (EI) at a maximal rate of k3 = 0.87 +/- 0.34 min-1. The inhibition is reversible (dissociation rate constant k4 = 0.038 +/- 0.002 min-1), requires the presence of the cofactors thiamin pyrophosphate and Mg2+, and is competitive with regard to pyruvate. The microscopic rate constants give a value of 5 nM for the overall dissociation constant [Ki = [E] [I]/[( EI] + [EI]) = Kik4/(k3 + k4)] compared with values of 10 and 3.5 nM obtained by steady-state methods. Thus acetylphosphinate binds by 5 orders of magnitude more tightly to pyruvate dehydrogenase than does pyruvate (Km = 0.35 mM). Acetylphosphinate also affects the pyruvate dehydrogenase complex fluorescence when excited at 290 nm in a time-dependent manner with a maximal rate constant of 0.99 min-1, suggesting a conformational change in the enzyme complex as the slow step in conversion of EI to EI (k3). All these features taken together suggest that the interaction of the pyruvate dehydrogenase with acetylphosphinate involves the formation of a thiamin pyrophosphate-acetylphosphinate adduct that resembles the normal reaction intermediate, 2-(1-carboxy-1-hydroxyethyl)thiamin pyrophosphate (alpha-lactylthiamin pyrophosphate).
TL;DR: Cooperative properties of KGD in the KGD complex are modulated by NADH and reducing 2 SH‐groups of K GD by dihydrolipoate leads to cooperativity in substrate binding.
TL;DR: It is suggested that NAD(+)-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.
Abstract: In this article we compare the kinetic behavior toward pyridine nucleotides (NAD+, NADH) of NAD+-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD+ was the varied substrate. However, the NAD+-linked malic enzyme exhibited the weakest affinity for NAD+ and the lowest sensitivity for NADH. It is suggested that NAD+-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.
TL;DR: It can be assumed that all three genes of the 2-oxoglutarate dehydrogenase multienzyme complex are transcribed as a single mRNA transcript under the control of a promoter, located more than 100 bp upstream of the E1o gene, analogous to the pyruvate dehydration complex in E. coli.
Abstract: The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. Previously the cloning in Escherichia coli of the gene encoding lipoamide dehydrogenase from A. vinelandii was reported [Westphal, A. H. & de Kok, A. (1988) Eur. J. Biochem. 172, 299–305]. The 3.2-kb fragment used for the sequence determination contained the main part of the gene encoding succinyltransferase. The complete E2o gene, as well as the gene encoding the 2-oxoglutarate dehydrogenase component, resided on a 14.7-kb fragment from which the 3.2-kb fragment was subcloned.
The protein-coding sequence of the gene consists of 1200 bp (400 codons, including the AUG start codon and the UGA stop codon). It is separated from the gene encoding the 2-oxoglutarate dehydrogenase component by 42 bp. No E. coli-like promoter sequence was found. A putative ribosome-binding site is located 9–15 bp upstream from the start codon. No terminator sequences were found downstream of the stop codon. This makes it likely that the three genes of the oxoglutarate dehydrogenase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogenase complex in E. coli. The intact gene was subcloned from the 14.7-kb fragment and brought to high expression under the influence of the vector-encoded lacZ promotor.
The similarity with the E. coli enzyme is high with 63% identity. Like the enzyme from E. coli, it consists of a single lipoyl-binding domain, a putative E1-and E3-binding domain and a catalytic domain. The main difference is found in a 31-residue sequence rich in alanine and proline located between the lipoyl domain and the putative E1- and E3-binding domain. This sequence, usually found in acetyltransferases and there identified as a highly mobile region by 1H-NMR, is replaced by a more polar, charged region in the E. coli enzyme.
Journal Article•
TL;DR: The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruVate, CoA and NAD, respectively.
Abstract: The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).
TL;DR: Data suggest that such autoantibodies, besides serving as diagnostic markers for primary biliary cirrhosis, may have a pathogenic role by their ability to inhibit important mitochondrial enzymes.
01 Jan 1990
TL;DR: Cultivation of the patient's fibroblasts in medium enriched with lipoic acid markedly improved these in vitro conversions of leucine and valine.
Abstract: Lactic acidosis and accumulation of 3-hydroxybutyrate and other citric acid cycle intermediates were found in an infant with a lethal syndrome of metabolic acidosis and renal tubular acidosis. Nevertheless, the patient was relatively well for 4 mo of life. The activity of the pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase, and branched-chain keto acid dehydrogenase were all reduced to levels 9 to 29% of control. In contrast, the activity of lipoamide dehydrogenase was normal. The conversion of 1-14C-leucine and 1-14C-valine to 14CO2 and of U-L-14C-valine to its major metabolic product 3-hydroxyisobutyric acid by fibroblasts derived from the patient was less than 5% of control. Cultivation of the patient's fibroblasts in medium enriched with lipoic acid markedly improved these in vitro conversions of leucine and valine.
TL;DR: 2-Oxoglutarate decarboxylase from the lactic acid bacterium Leuconostoc oenos was purified by precipitation with PEG and ion-exchange chromatography and showed significant similarities to the corresponding mitochondrial enzyme of Euglena gracilis.
Abstract: Summary: 2-Oxoglutarate decarboxylase from the lactic acid bacterium Leuconostoc oenos was purified by precipitation with PEG and ion-exchange chromatography. The strictly thiamin-pyrophosphate-dependent enzyme decarboxylated 2-oxoglutarate to succinic semialdehyde. Oxalacetate was metabolized to a lesser extent. The measured K
m values for 2-oxoglutarate and thiamin pyrophosphate were 1 mM and 0·03 mM respectively. The enzyme had a molecular mass in the range 65-70 kDa, did not consist of subunits and showed significant similarities to the corresponding mitochondrial enzyme of Euglena gracilis.
TL;DR: The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.
TL;DR: Peptide maps of purified OGC show structural differences between the carrier from heart and liver of the same animal species, indicating the existence of isoforms of the OGC in heart and Liver.
TL;DR: The findings suggest that several denitrifying Pseudomonas strains contained at least two lipoamide dehydrogenases, one NAD+-specific, the other NADP+- Specific, if the cells were grown anaerobically with aromatic compounds.
Abstract: Several denitrifying Pseudomonas strains contained an NADP+-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD+-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD+-specificity. The reaction stoichiometry and the apparent Km-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD+ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP+ 0.03 mM. Isocitrate dehydrogenase was NADP+-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD+-specific, the other NADP+-specific.
TL;DR: Rotational mobility of the lipoyl domain of a number of 2-oxoacid dehydrogenase complexes was investigated by transient dichroism after the domain had been specifically labeled with the triplet probe eosin-5-maleimide, finding a surprisingly weak dependence on viscosity.
Abstract: Rotational mobility of the lipoyl domain of a number of 2-oxoacid dehydrogenase complexes was investigated by transient dichroism after the domain had been specifically labeled with the triplet probe eosin-5-maleimide. Complexes investigated included pyruvate dehydrogenase complexes from Bacillus stearothermophilus, ox heart, and Escherichia coli (in which the E2 component had been genetically engineered to contain one lipoyl domain) and 2-oxoglutarate dehydrogenase complexes from ox heart and E. coli. Measurements were also performed with ox heart pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes specifically labeled on E1. Anisotropy decays were recorded in glycerol-buffer solutions of varying viscosity and at different temperatures. For E2-labeled complexes, the decays were found to be multiexponential, and the fastest correlation time was considerably shorter than expected for tumbling of the whole complex. This fast correlation time was absent from E1-labeled complexes and was assigned to independent motion of the lipoyl domain. Plots of the fast correlation time against eta/T showed a surprisingly weak dependence on viscosity and extrapolated to a time of 30-40 microseconds at zero viscosity. To explain this result, a model is proposed in which the lipoyl domain is in equilibrium between "free" and bound states. The time of 30-40 microseconds is shown to correspond to 1/koff, where koff is the rate constant for dissociation of the domain from binding sites on the complex. This dissociation phenomenon only contributes to the anisotropy decay when the viscosity of the solution is sufficiently high to slow the tumbling of the whole complex to times that are long in comparison to 1/koff.
TL;DR: It is suggested that the hyperammonemic state induced by MSI might derange the operation of the malate-aspartate shuttle and the existence of a small population of mitochondria which was highly vulnerable either to ammonia or to MSI.