Interaction of octanoate with branched-chain 2-oxo acid oxidation in rat and human muscle in vitro

Acetate and butanoate inhibited and hexanoate and octanoate increased the 14CO2 production from 0.1 mM [1-14C]-labelled 2-oxoisocaproate (KIC) and 2-oxoisovalerate (KIV) in rat hemidiaphragms. Octanoate increased KIC and KIV oxidation in rat soleus muscle, too, inhibited it in human skeletal muscle and had a divergent effect in rat and human heart slices. In rat hemidiaphragms octanoate primarily affected the process of oxidative decarboxylation. No effect was found on transamination rates of branched-chain amino acids and on the CO2 production beyond alpha-decarboxylation. The reverse transamination of branched-chain 2-oxo acids and their incorporation into protein decreased in the presence of octanoate. Octanoate had no effect on KIC and KIV oxidation at higher 2-oxo acid concentrations and in hemidiaphragms from 3-day-starved rats. The observed interactions are discussed and related to regulatory mechanisms, which are known to affect the branched-chain 2-oxo acid dehydrogenase complex.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Inhibition of glycine synthase by branched-chain α-keto acids: A possible mechanism for abnormal glycine metabolism in ketotic hyperglycinemia

The effect of various amino acid metabolites on glycine oxidation by rat liver homogenate was investigated. Three compounds, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-β-methylvaleric acid, were found to inhibit glycine oxidation by 40–60%. In addition, these compounds also inhibited the glycine-CO₂ exchange reaction, a partial reaction of glycine synthase. The reverse reaction, glycine synthesis, was stimulated 4-fold by these α-keto acids. Pyruvate and α-ketoglutarate had no effect on any of these reactions. The parent amino acids, valine, isoleucine, and leucine, also had no effect on the reactions nor did any of their other metabolites with the exception of the branched-chain α-keto acids. The concentration dependence of the inhibition of glycine oxidation and stimulation of glycine synthesis by these branched-chain α-keto acids suggested that the inhibition of glycine oxidation by these compounds was the result of their further oxidation by branched-chain α-keto acid dehydrogenase. However, the products of the branched-chain α-keto acid dehydrogenase, isobutyryl CoA, isovaleryl CoA, or α-methylbutyryl CoA had no effect on glycine oxidation. Thus, it appeared that either the branched-chain α-keto acids altered glycine oxidation by direct binding to glycine synthase or that electrons derived from the oxidation of branched-chain α-keto acids were transferred to the glycine synthase system. It is proposed that glycine synthase and branched-chain α-keto acid dehydrogenase either share a common subunit, possibly lipoamide dehydrogenase, or are so arranged on the mitochondrial membrane that electron transfer between these two enzymes occurs.     

The effect of octanoate and palmitate on the metabolism of valine in perfused hindquarter of rat.

The effect of octanoate and palmitate on the oxidation of 14C-labelled valine has been studied in perfused hindquarter of rat. 1. The oxidation rate of valine increases 30 times when the concentration of valine is increased from 0.1 mM to 5 mM. 2. Octanoate at a 5 mM concentration effected a 10-fold increase in the flux through the alpha-ketoisovalerate dehydrogenase step and a 5-fold increase at 0.5 mM concentration. 3. Palmitate (1 mM) effects only a moderate increase in the valine oxidation. 4. With no octanoate there was a great accumulation of alpha-ketoisovalerate in both the muscle and the perfusion medium. 5. With octanoate little alpha-ketoisovalerate accumulated whereas 3-hydroxyisobutyrate was found in high concentration both in the muscle and in the medium. 6. Octanoate stimulated the production of citric-acid-cycle intermediates and lactate. 7. The results are discussed in relation to valine metabolism in the body.     

Regulatory effects of fatty acids on decarboxylation of leucine and 4-methyl-2-oxopentanoate in the perfused rat heart.

The regulatory effects of fatty acids on the oxidative decarboxylation of leucine and 4-methyl-2-oxopentanoate were investigated in the isolated rat heart. Infusion of the long-chain fatty acid palmitate resulted in both an inactivation of the branched-chain 2-oxo acid dehydrogenase and an inhibition of the measured metabolic flux through this enzyme complex. Pyruvate addition also caused both an inactivation and an inhibition of the flux through the complex. On the other hand, the medium-chain fatty acid octanoate caused an activation of and a stimulation of flux through the branched-chain 2-oxo acid dehydrogenase when the perfusion conditions before octanoate addition maintained the enzyme complex in its inactive state. When the enzyme complex was activated before octanoate infusion, this fatty acid caused a significant inhibition of the flux through the branched-chain 2-oxo acid dehydrogenase reaction. Inclusion of glucose in the perfusion medium prevented the octanoate-mediated activation of the branched-chain 2-oxo acid dehydrogenase.     

Regulation of branched-chain amino acid oxidation in isolated muscles, nerves and aortas of rats.

1. The oxidation of the three branched-chain amino acids was regulated in parallel fashion in rat tissues studied in vitro. 2. With 0.1 mM-[1-14C]isoleucine as substrate in the presence of 5.5 mM-glucose, 14CO2 production was 0.6 mumol/2 h per g in the aorta, 0.3 in peripheral nerve, 0.2 in muscle and 0.13 in spinal cord. 3. The ratio 14C oxidized/14C incorporated into proteins with 0.1 mM-[1-14C]leucine was 1.3 in hemidiaphragms, 3.3 in sciatic nerve and 1.0 in nerves undergoing Wallerian degeneration. Leucine oxidation decreased only slightly during degeneration, but protein synthesis doubled. 4. Hemidiaphragms incubated with [1-14C]leucine or 4-methyl-2-oxo[1-14C]pentanoate increased 14CO2 production 7-9-fold as substrate concentration was increased from 0.1 to 0.5 mM; under the same conditions 14CO2 production by nerves increased only 2-3-fold. 5. 2-Oxoglutarate stimulated the oxidation of the branched-chain amino acids by muscles and peripheral nerves and the oxidation of 4-methyl-2-oxopentanoate by hemidiaphragms but not by nerves. 6. Octanoate (0.1-1.0 mM) markedly stimulated the oxidation of branched-chain amino acids and of 4-methyl-2-oxopentanoate in hemidiaphragms, but inhibited oxidation of both by peripheral nerves and spinal cord. In aortas, oxidation of isoleucine (the only substance tested) was inhibited by octanoate. 7. The effects of octanoate and 2-oxoglutarate on leucine oxidation by hemidiaphragms were additive at low concentrations. When maximally stimulating concentrations of either agent were used, addition of the other was ineffective. 8. Pyruvate inhibited the oxidation of branched-chain amino acids and 4-methyl-2-oxopentanoate in all tissues tested. 9. Insulin did not affect the oxidation of 4-methyl-2-oxopentanoate by muscles or nerves. 10. The oxidative decarboxylation of the branched-chain alpha-oxo acids is suggested as a regulatory site of branched-chain amino acid oxidation. Differences in regulation between muscle on the one hand, and nerve and aorta on the other, are discussed.     

Branched-chain amino acid (BCAA) supplementation enhances adaptability to exercise training of mice with a muscle-specific defect in the control of BCAA catabolism

Branched-chain α-keto acid dehydrogenase (BCKDH) kinase (BDK) suppresses the branched-chain amino acid (BCAA) catabolism by inactivation of the BCKDH complex. The muscle-specific BDK-deficient (BDK-mKO) mice showed accelerated BCAA oxidation in muscle and decreased endurance capacity after training (Xu et al. PLoS One. 12 (2017) e0180989). We here report that BCAA supplementation overcompensated endurance capacity in BDK-mKO mice after training.     

Branched-chain Amino Acid Metabolon: INTERACTION OF GLUTAMATE DEHYDROGENASE WITH THE MITOCHONDRIAL BRANCHED-CHAIN AMINOTRANSFERASE (BCATm)*

The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain α-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain α-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5′-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5′-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5′-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

Role of Branched-Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism

Abstract: Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α-ketoglutarate to form the branched-chain α-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-¹⁴C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.     

Mechanism of increased conversion of branched chain keto acid dehydrogenase from inactive to active form by a medium chain fatty acid (octanoate) in skeletal muscle.

We and others have previously shown that octanoate increases the oxidation of branched chain amino acids (BCAA) in skeletal muscle. The present study was designed to investigate the mechanism of this increased oxidation. Studies were performed with rat hind limbs perfused with 0.50 mM L-[1-14C]leucine with or without octanoate. The flux through branched chain keto acid (BCKA) dehydrogenase was measured, and the basal and total activity of BCKA dehydrogenase in skeletal muscle was determined. The rate of flux through BCKA dehydrogenase increased by 37, 119, and 297% with 0.5, 1.0, and 2.0 mM octanoate, respectively. This increase in flux was not due to a change in BCAA aminotransferase activity but was due to an increase in the basal activity of BCKA dehydrogenase. There was a strong correlation (r = 0.96) between increases in flux through BCKA dehydrogenase and increases in the basal activities of BCKA dehydrogenase. Preincubation of BCKA dehydrogenase with Mg2+ caused full activation of this enzyme, but preincubation with octanoate did not activate this enzyme. On the other hand, octanoate completely prevented the ATP-dependent inactivation of fully activated BCKA dehydrogenase. We conclude that octanoate increases the oxidation of leucine in skeletal muscle by increasing the activation of BCKA dehydrogenase. The mechanism of this activation is the inhibition of BCKA dehydrogenase kinase rather than the stimulation of a specific or nonspecific protein phosphatase.     

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.     

Branched-chain amino acid oxidation by isolated rat tissue preparations

Branched-chain amino acid transaminase activity, branched-chain α-keto acid dehydrogenase activity, and leucine oxidation were measured in homogenates and slices of several rat tissues. Transaminase activity was highest in heart, while dehydrogenase activity was highest in liver. Leucine oxidation in isolated tissues may be limited by either transaminase or dehydrogenase activity depending upon the relative activities of these two enzymes in the tissue. The results suggest that, as the load of branched-chain amino acids increases, the liver may become an increasingly important site for the degradation of branched-chain α-keto acids.     

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways

Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5′-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5′-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.     

The effect of acetate on the regulation of the branched chain α-keto acid and the pyruvate dehydrogenase complexes in the perfused rat liver

The regulatory consequences of acetate infusion on the pyruvate and the branched chain α-keto acid dehydrogenase reactions in the isolated, perfused rat liver were investigated. Metabolic flux through these two decarboxylation reactions was monitored by measuring the rate of ¹⁴CO₂ production from infused 1-¹⁴C-labeled substrates. When acetate was presented to the liver as the sole substrate the rate of ketogenesis which resulted was maximal at concentrations of acetate in excess of 10 mm. The increase in hepatic ketogenesis during acetate infusion was not accompanied by an alteration of the mitochondrial oxidation-reduction state as measured by the ratio of β-hydroxybutyrate/ acetoacetate in the effluent perfusate. While acetate infusion did not affect the rate of α-keto[1-¹⁴C]isocaproate decarboxylation, the rate of α-keto[1-¹⁴C]isovalerate decarboxylation was stimulated appreciably upon acetate addition. No change was observed in the amount of extractable branched chain α-keto acid dehydrogenase during acetate infusion. The rate of [1-¹⁴C]pyruvate decarboxylation was stimulated in the presence of acetate at low (<1 mm) but not at high (>1 mm) perfusate pyruvate concentrations. The stimulation of the metabolic flux through the pyruvate dehydrogenase reaction upon acetate infusion was accompanied by an increase in the activation state of the pyruvate dehydrogenase complex from 25.7 to 35.6% in the active form. In a liver perfused in the presence of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, at a low concentration of pyruvate (0.05 mm) the infusion of acetate did not affect the rate of pyruvate decarboxylation. As the rate of mitochondrial acetoacetate efflux is increased during acetate infusion the stimulation of pyruvate and α-ketoisovalerate decarboxylation is attributed to an accelerated rate of exchange of mitochondrial acetoacetate for cytosolic pyruvate or α-ketoisovalerate on the monocarboxylate transporter.     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli.

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.     

Mitochondrial 2-oxoacid dehydrogenase complexes of animal tissues

The pyruvate dehydrogenase and branched-chain 2-oxoacid dehydrogenase complexes of animal mitochondria are inactivated by phosphorylation of serine residues, and reactivated by dephosphorylation. In addition, phosphorylated branched-chain complex is reactivated, apparently without dephosphorylation, by a protein or protein-associated factor present in liver and kidney mitochondria but not in heart or skeletal muscle mitochondria. Interconversion of the branched-chain complex may adjust the degradation of branched-chain amino acids in different tissues in response to supply. Phosphorylation is inhibited by branched-chain ketoacids, ADP and TPP. The pyruvate dehydrogenase complex is almost totally inactivated (99%) by starvation or diabetes, the kinase reactions being accelerated by products of fatty acid oxidation and by a protein or protein-associated factor induced by starvation or diabetes. There are three sites of phosphorylation, but only sites 1 and 2 are inactivating. Site 1 phosphorylation accounts for 98% of inactivation except during dephosphorylation when its contribution falls to 93%. Sites 2 and 3 are only fully phosphorylated when the complex is fully inactivated (starvation, diabetes). Phosphorylation of sites 2 and 3 inhibits reactivation by phosphatase. The phosphatase reaction is activated by Ca2+ (which may mediate effects of muscle work) and possibly by uncharacterized factors mediating insulin action in adipocytes.