Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex.
ABSTRACT: Highly purified branched-chain 2-oxo acid dehydrogenase complex (BCOADC) oxidizes 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with Km values of 67 microM and 18 microM respectively. The Vmax. for oxidation of these substrates is 27% and 53% respectively of that for 3-methyl-2-oxobutyrate. Highly purified pyruvate dehydrogenase complex (PDC) oxidizes 2-oxobutyrate (Km 100 microM; Vmax. 49% of that for pyruvate) but not 4-methylthio-2-oxobutyrate, whereas 2-oxoglutarate dehydrogenase complex will not utilize either 2-oxo acid as substrate. BCOADC kinase is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with half-maximal inhibition by 45 microM and 50 microM respectively. Phosphorylation of BCOADC in isolated adipocytes is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, consistent with their inhibitory action of BCOADC kinase. Phosphorylation of PDC is decreased by 2-oxobutyrate, but not by 4-methylthio-2-oxobutyrate.
Project description: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.
Project description: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.
Project description:The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.
Project description:2-Oxobutyrate (2-OBA), as a toxic metabolic intermediate, generally arrests the cell growth of most microorganisms and blocks the biosynthesis of target metabolites. In this study, we demonstrated that using the acetate bypass to replace the pyruvate dehydrogenase complex (PDHc) in <i>Escherichia coli</i> could recharge the intracellular acetyl-CoA pool to alleviate the metabolic toxicity of 2-OBA. Furthermore, based on the crystal structure of pyruvate oxidase (PoxB), two candidate residues in the substrate-binding pocket of PoxB were predicted by computational simulation. Site-directed saturation mutagenesis was performed to attenuate 2-OBA-binding affinity, and one of the variants, PoxB<sup>F112W</sup>, exhibited a 20-fold activity ratio of pyruvate/2-OBA in substrate selectivity. PoxB<sup>F112W</sup> was employed to remodel the acetate bypass in <i>E. coli</i>, resulting in l-threonine (a precursor of 2-OBA) biosynthesis with minimal inhibition from 2-OBA. After metabolic detoxification of 2-OBA, the supplies of intracellular acetyl-CoA and NADPH (nicotinamide adenine dinucleotide phosphate) used for l-threonine biosynthesis were restored. Therefore, 2-OBA is the substitute for pyruvate to engage in enzymatic reactions and disturbs pyruvate metabolism. Our study makes a straightforward explanation of the 2-OBA toxicity mechanism and gives an effective approach for its metabolic detoxification.
Project description:A radiochemical assay was developed to measure pyruvate dehydrogenase complex (PDC) activity in liver and heart without interference by branched-chain 2-oxo acid dehydrogenase (BCODH). Decarboxylation of pyruvate by BCODH was eliminated by using low pyruvate concentration (0.5 mM), a preferred substrate for BCODH (3-methyl-2-oxopentanoate) that is not used by PDC, and a competitive inhibitor of BCODH, dichloroacetate. This method was validated by assaying a combination of both purified enzymes and tissue homogenates with known amounts of added BCODH. The actual percentage of active PDC decreased after 48 h starvation from 13.6 to 3.1 in liver and from 77.1 to 9.0 in heart. Total PDC activity (munits of PDC/units of citrate synthase) in starved rats was increased by 34% in liver and decreased by 23% in heart. Total PDC activity (munits/g wet wt.) in fed- and starved-rat liver was 0.8 and 1.3, and in heart was 6.6 and 5.8, respectively.
Project description:The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of beta-isopropylmalate, beta-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.
Project description:The relation between pyruvate utilization and acetylcholine synthesis was investigated in minces of adult rat brain. The flux of pyruvate to acetylcholine was less than 1% of that to CO2; nevertheless, a number of agents which inhibited conversion of [1-14C]-pyruvate or [2-14C]pyruvate into 14CO2 were associated with corresponding decreases in the conversion of [2-14C]pyruvate into acetylcholine. The amount of acetylcholine produced by minces of whole rat brain, measured by g.l.c.-mass spectrometry, decreased similarly. Among the inhibitory compounds tested were 3-bromopyruvate, an irreversible inhibitor of pyruvate dehydrogenase; 2-oxobutyrate, a competitive inhibitor of pyruvate dehydrogenase; other 2-oxo acids; and amobarbital and pentobarbital. Linear-regression equations relating CO2 production to acetylcholine synthesis gave correlation coefficients of 0.89-0.93 for the combined observations. The inhibition of acetylcholine synthesis could not be attributed to inhibition of choline acetyltransferase. Incorporation of [2-14C]pyruvate into lipids, proteins and nucleic acids was effected less than that into acetylcholine. Under these experimental conditions, it was shown that pyruvate utilization can limit acetylcholine synthesis.
Project description:The effects of aliphatic 2-oxocarboxylic acids, at concentrations of up to 40mm, on the function of pancreatic islets from ob/ob (obese-hyperglycaemic) mice were investigated. 1. 2-Oxopentanoate, dl-3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate and 2-oxohexanoate all induced insulin release by isolated incubated islets and a biphasic insulin-secretory pattern in perfused mouse pancreas. The last two substances were similar in potency to glucose. Pyruvate, 2-oxobutyrate, 3-methyl-2-oxobutyrate and 2-oxo-octanoate did not induce insulin release significantly. 2. 2-Oxocarboxylic acids with significant insulin-secretory potency also induced significant (45)Ca uptake by isolated incubated islets. 3. The rates of decarboxylation of [1-(14)C]pyruvate, 3-methyl-2-oxo[1-(14)C]butyrate and 4-methyl-2-oxo[1-(14)C]pentanoate were twice as high as the rates of oxidation of the corresponding U-(14)C-labelled compounds. However, whereas the rates of metabolism of labelled pyruvate and 3-methyl-2-oxobutyrate steadily increased over the concentration range 1-40mm, those of labelled 4-methyl-2-oxopentanoate and d-[U-(14)C]glucose levelled off at concentrations above 10mm. 4. Omission of (40)CaCl(2) from the incubation medium reduced the rate of oxidation of the insulin secretagogue [U-(14)C]4-methyl-2-oxopentanoate, but left that of the non-(insulin secretagogue) [U-(14)C]3-methyl-2-oxobutyrate unaffected. 5. Only glucose, and not pyruvate, 3-methyl-2-oxobutyrate and 4-methyl-2-oxopentanoate, significantly inhibited oxidation of endogenous fatty acids. 6. It is suggested that stimulus-secretion coupling and the resulting exocytosis of insulin in pancreatic beta-cells may modulate both fuel oxidation and (45)Ca uptake.
Project description:The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney. A broad pH optimum around 7.8, similar dependence on ionic strength, and independence of the nature of the buffer anions or cations characterized preparations from both tissues. Michaelis constants for the binding of pyruvate, thiamin pyrophosphate, NAD(+) and CoA were also similar. Enzyme from both tissues was inhibited by NADH, by copper and other heavy metals, by high concentrations of tricarboxylic acid-cycle intermediates, and by preincubation with ATP. Acetyl-CoA itself did not appear to inhibit these preparations, although some commercial preparations of acetyl-CoA did contain an inhibitor. Although oxaloacetate and alpha-oxobutyrate were weak inhibitors, a number of other alpha-oxo acids including phenylpyruvate did not inhibit. The properties of the pyruvate dehydrogenase complex from brain and kidney appeared similar.
Project description:The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 188.8.131.52 and EC 184.108.40.206) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively.