2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase activities in plant mitochondria: interaction via a common coenzyme a pool

2-Oxoglutarate (2-OG)-dependent O2 uptake by washed or purified turnip (Brassica rapa L.) and pea (Pisum sativum L. cv. Massey Gem) leaf mitochondria, in the presence of malonate, was inhibited between 65 and 90% by micromolar levels of pyruvate. The inhibition was not observed in the absence of malonate and was reversed by alpha-cyano-4-hydroxycinnamic acid. The inhibition was also reversed by oxaloacetate or by malate, but not by any other tricarboxylic acid cycle intermediates. The stimulation of O2 uptake by oxaloacetate was half maximal at 8-9 microM and was transient, indicating its action was not mediated through the complete metabolic removal of pyruvate. Pyruvate had not effect on 2-OG oxidation under conditions in which pyruvate dehydrogenase was not active, indicating that pyruvate metabolism, rather than pyruvate itself, was responsible for producing the inhibition of 2-OG oxidation. Similar results were obtained with detergent-treated mitochondrial extracts with the exception that the inhibition of 2-OG oxidation by pyruvate could also be reversed by coenzyme A. The results suggest that pyruvate inhibits 2-oxoglutarate oxidation, in intact plant mitochondria, by sequestering intramitochondrial CoA as acetyl-CoA and, in the absence of citrate synthase activity, reduces the amount of free coenzyme A available for 2-oxoglutarate dehydrogenase. These results indicate that pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase share a common CoA pool within plant mitochondria and that the turnover of the acyl-CoA product of one enzyme will dramatically influence the activity of the other.     

2-Oxoglutarate decarboxylase ofLeuconostoc oenos

InLeuconostoc oenos, the typical organism of the malolactic fermentation of wine, a 2-oxoglutarate decarboxylase was detected. This inducible enzyme decarboxylates 2-oxoglutarate but not pyruvate. The resulting succinaldehydate is rapidly reduced to 4-hydroxybutyrate or oxidized to succinate in further reactions. 2-Oxoglutarate decarboxylase is thiamin-diphosphate-dependent; the pH optimum is at 5.3 and theK _m value for 2-oxoglutarate is 1.8 mmol/L.     

2-Oxoglutarate transport system in Staphylococcus aureus

2-[(14)C]oxoglutarate uptake in resting cells of Staphylococcus aureus 17810S occurs via two kinetically different systems: (1) a secondary, electrogenic 2-oxoglutarate:H(+) symporter (K(m)=0.105 mM), energized by an electrochemical proton potential (Delta mu H(+)) that is generated by the oxidation of endogenous amino acids and sensitive to ionophores, and (2) a Delta mu H(+)-independent facilitated diffusion system (K(m)=1.31 mM). The 2-oxoglutarate transport system of S. aureus 17810S can be classified as a new member of the MHS (metabolite:H(+) symporter) family. This transporter takes up various dicarboxylic acids in the order of affinity: succinate = malate > fumarate > 2-oxoglutarate > glutamate. Energy conservation with 2-oxoglutarate was studied in starved cells of strain 17810S. Initial transport of 2-oxoglutarate in these cells is energized by Delta mu H(+) generated via hydrolysis of residual ATP. Subsequent oxidation of the accumulated 2-oxoglutarate generates Delta mu H(+) for further, autoenergized transport of this 2-oxoacid and also for Delta mu H(+)-linked resynthesis of ATP. In the cadmium-sensitive S. aureus 17810S, Cd(2+) accumulation strongly inhibits energy conservation with 2-oxoglutarate at the level of Delta mu H(+) generation, without direct blocking of the 2-oxoglutarate transport system or ATP synthase complex. In the cadmium-resistant S. aureus 17810R, Cd(2+) does not affect energy conservation due to its extrusion by the Cd(2+) efflux system (Cd(2+)-ATPase of P-type), which prevents Cd(2+) accumulation.     

2-Oxoglutarate analogue inhibitors of prolyl hydroxylase domain 2

Analogues of the 2-oxoglutarate cosubstrate of the human oxygen sensing enzyme prolyl hydroxylase domain 2 (PHD2) with variations in the potential iron-chelating group were screened as inhibitors and for binding (using non-denaturing electrospray ionization mass spectrometry) to PHD2.     

Affinity and stability modifications of immobilized alcohol dehydrogenase through multipoint copolymerization

Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), a potentially useful enzyme for cofactor regeneration processes, was covalently immobilized in a multipoint fashion by activation with acryloyl chloride and subsequent copolymerization in a polyacrylamide gel. Several properties such as the activity and stability were systematically studied for the free enzyme, the acryloate-enzyme and the immobilized enzyme. The activation energy was significantly lowered upon immobilization. The thermal stability of the immobilized enzyme was, however, greatly increased. But its maximum activity was observed at a lower temperature. These results suggest an important effect of the diffusional restrictions and of the mode of activation and immobilization on the activity and the stability of the enzyme.     

Metabolic control analysis of eucaryotic pyruvate dehydrogenase multienzyme complex

Metabolic control analysis (MCA) of pyruvate dehydrogenase multienzyme (PDH) complex of eucaryotic cells has been carried out using both in vitro and in vivo mechanistic models. Flux control coefficients (FCC) for the sensitivity of pyruvate decarboxylation rate to activities of various PDH complex reactions are determined. FCCs are shown to be strong functions of both pyruvate levels and various components of PDH complex. With the in vitro model, FCCs are shown to be sensitive to only the E1 component of the PDH complex at low pyruvate concentrations. At high pyruvate concentrations, the control is shared by all of the components, with E1 having a negative influence while the other three components, E2, X, and K, exert a positive control over the pyruvate decarboxylation rate. An unusual behavior of deactivation of the E1 component leading to higher net PDH activity is shown to be linked to the combined effect of protein X acylation and E1 deactivation. The steady-state analysis of the in vivo model reveals multiple steady state behavior of pyruvate metabolism with two stable and one unstable steady-states branches. FCCs also display multiplicity, showing completely different control distribution exerted by pyruvate and PDH components on three branches. At low pyruvate concentrations, pyruvate supply dominates the decarboxylation rate and PDH components do not exert any significant control. Reverse control distribution is observed at high pyruvate concentration. The effect of dilution due to cell growth on pyruvate metabolism is investigated in detail. While pyruvate dilution effects are shown to be negligible under all conditions, significant PDH complex dilution effects are observed under certain conditions. Comparison of in vitro and in vivo models shows that PDH components exert different degrees of control outside and inside the cells. At high pyruvate levels, PDH components are shown to exert a higher degree of control when reactions are taking place inside the cells as compared to the in vitro situation.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Quantitative control analysis of branched-chain 2-oxo acid dehydrogenase complex activity by feedback inhibition.

The potential for branched-chain 2-oxo acid dehydrogenase complex (BCOADC) activity to be controlled by feedback inhibition was investigated by calculating the Elasticity Coefficients for several feedback inhibitors. We suggest that feedback inhibition is a quantitatively important regulatory mechanism by which branched-chain 2-oxo acid dehydrogenase activity is regulated. The potential for control of enzyme activity is greater for NADH than for the acyl-CoA products, and suggests that factors that alter the redox potential may physiologically regulate BCOADC activity through a feedback inhibitory mechanism in vivo. Local pH may also be an important regulatory control factor.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

The 2-oxoacid dehydrogenase multienzyme complex of Haloferax volcanii

Those aerobic archaea whose genomes have been sequenced possess four adjacent genes that, by sequence comparisons with bacteria and eukarya, appear to encode the component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the archaea. In Thermoplasma acidophilum, evidence has been presented that the heterologously expressed recombinant enzyme possesses activity with the branched chain 2-oxoacids and, to a lesser extent, with pyruvate. In the current paper, we demonstrate that in Haloferax volcanii the four genes are transcribed as an operon in vivo. However, no functional complex or individual enzyme, except for the dihydrolipoamide dehydrogenase component, could be detected in this halophile grown on a variety of carbon sources. Dihydrolipoamide dehydrogenase is present at low catalytic activities, the level of which is increased three to fourfold when Haloferax volcanii is grown on the branched-chain amino acids valine, leucine and isoleucine.     

Multi-site phosphorylation in ox-kidney branched-chain 2-oxoacid dehydrogenase complex

Tryptic [³²P]phosphopeptides were prepared from [³²P]phosphorylated ox-kidney branched-chain complex and analysed by high-voltage paper electrophoresis at pH 1.9. In the maximally phosphorylated complex 3 tryptic [³²P]phosphopeptides were identified (TA, TB, TC). R_F-values relative to N⁶-dinitrophenyllysine were (mean ± SEM for 25 obs.): TA, 1.53 ± 0.03; TB, 1.07 ± 0.02; TC, 0.65 ± 0.01. Relative rates of phosphorylation were TA> TB> TC. Phosphorylation of TA reached a maximum when about 66% of the complex was inactivated. Phosphorylation of TB and TC was associated mainly with 66–95% inactivation of the complex.     

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.     

Cloning and characterization of the Alcaligenes eutrophus 2-oxoglutarate dehydrogenase complex

Abstract Nucleotide sequence analysis of a 3.3-kb genomic Eco RI fragment and of relevant subfragments of a genomic 13.2-kb Sma I fragment of Alcaligenes eutrophus , which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative −35 / −10 promoter in the order odhA, odhB , and odhL . In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous expression of the A. eutrophus odh genes in E. coli XL1-Blue and in the kgdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.     

Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.