Presence Of Α-ketoglutarate Research Articles

Inhibition of Pyruvate Carboxylation by Fluorocitrate in Rat Kidney Mitochondria

Rat kidney mitochondria, in the presence of ATP, Pi, and magnesium ions, converted pyruvate and bicarbonate to malate, fumarate, and citrate and, in the presence of glutamate, also to aspartate. The addition of fluorocitrate strongly inhibited pyruvate utilization and 14CO2 incorporation into organic acids. The inhibition of 14CO2 incorporation into organic acids in the presence of fluorocitrate appeared to be caused by malonyl coenzyme A produced by fluorocitrate stimulation of acetyl-CoA carboxylase. This inhibition by fluorocitrate of 14CO2 incorporation was reversed by carnitine, acetylcarnitine, acylcarnitines, isocitrate, α-ketoglutarate, succinate, fumarate, and glutamate, but not by citrate, β-hydroxybutyrate, or caprylate. The ratio of carnitine to fluorocitrate determined the degree of reversal of this inhibition. The reversal of inhibition by carnitine or acylcarnitine resulted in greatly increased citrate formation, with nearly all of the 14CO2 being incorporated into citrate, and in nearly complete inhibition of malate synthesis. Fluorocitrate inhibited oxidation of citrate. In the presence of tricarboxylic cycle intermediates, the incorporation of bicarbonate-14C into malate was greatly increased. With fluorocitrate in the system, the incorporation of radioactive bicarbonate into malate in the presence of α-ketoglutarate and succinate did not change, but decreased in the presence of fumarate.

The biosynthesis of proline by Tetrahymena pyriformis

1. 1. This report concerns the pathway for proline formation in Tetrahymena pyriformis and the biochemical basis for the sparing effects of citrulline, ornithine, and proline on the arginine requirement of this organism. Proline is formed from arginine through the intermediates citrulline, ornithine, and glutamate γ-semialdehyde ( Δ 1-pyrroline-5-carboxylate). 2. 2. The first step of this pathway is catalyzed by arginine deiminase ( L-arginine iminohydrolase, EC 3.5.3.6). This enzyme was purified 75-fold and was found to be strongly inhibited by L-ornithine. The inhibition provides a means of control of ornithine formation in the cell. 3. 3. Citrulline hydrolase, which cleaves citrulline to form ornithine, was purified 79-fold. The enzyme has interacting substrate sites, but it serves no known function in the control of proline formation. 4. 4. Ornithine, in the presence of α-ketoglutarate, is converted to glutamate γ-semialdehyde by ornithine δ-transaminase ( L-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13). This enzymatic activity is located in particulate fractions of the cell and is moderately inhibited by L-valine but not by proline. It was purified 105-fold. 5. 5. Glutamate γ-semialdehyde is converted to proline by Δ 1-pyrroline-5-carboxylate reductase ( L-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2), which was purified 38-fold.

Metabolism of lysine in rat liver

It was shown previously that lysine was degraded in the mitochondria in the presence of α-ketoglutarate and, under these conditions, saccharopine accumulated (1). The obligatory requirement of α-ketoglutarate for lysine breakdown as well as for saccharopine formation suggest that lysine is metabolized, in mammalian liver, by a reversal of reaction sequence described for its biosynthesis in yeast (2–6). The present report describes that saccharopine is further metabolized to α-aminoadipic acid in the mitochondria and that NAD rather than NADP is required in this reaction.

Utilization of acetate by Beggiatoa.

Burton, Sheril D. (Institute of Marine Science, University of Alaska, College), Richard Y. Morita, and Wayne Miller. Utilization of acetate by Beggiatoa. J. Bacteriol. 91:1192-1200. 1966.-A proposed system which would permit acetate incorporation into four-carbon compounds without the presence of key enzymes of the citric acid cycle or glyoxylate cycle is described. In this system, acetyl-coenzyme A (CoA) is condensed with glyoxylate to form malate, which, in turn, is converted to oxaloacetate. Oxaloacetate then reacts with glutamate to produce alpha-ketoglutarate, which is subsequently converted to isocitrate. Cleavage of isocitrate produces glyoxylate and succinate. Thus, the proposed system is similar to the glyoxylate bypass in that malate is produced from glyoxylate and acetyl-CoA, but differs from both the citric acid cycle and the glyoxylate bypass, since citrate and fumarate are not involved. Fumarase, aconitase, catalase, citritase, pyruvate kinase, enolase, phosphoenolpyruvate carboxylase, lactic dehydrogenase, alpha-ketoglutarate dehydrogenase, and condensing enzyme were not detectable in crude extracts of Beggiatoa. Succinate was oxidized by a soluble enzyme not associated with an electron-transport particle. Isocitrate was identified as the sole compound labeled when C(14)O(2) was added to a reduced nicotinamide adenine dinucleotide, CO(2) generating system (crystalline glucose-6-phosphate dehydrogenase and glucose-6-phosphate) in the presence of alpha-ketoglutarate.

AN AROMATIC AMINO ACID TRANSAMINASE FROM MUNG BEAN

A transaminase has been isolated and purified from young mung bean plants (Phaseolus aureus Roxb.). The enzyme catalyzes the transamination of phenylalanine in the presence of α-ketoglutarate with the production of equimolar amounts of phenylpyruvate and glutamate. Tyrosine and tryptophan also serve as substrates, and relative rate measurements indicate that only one enzyme is involved. In addition to α-ketoglutarate the enzyme also utilizes pyruvate, and to some extent glyoxylate and oxaloacetate as amino acceptors. The enzyme is stable in solution at 0–4 °C for several weeks, and acetone powders of the young plants stored at 0–4 °C retained their activity for several months. The enzyme is inhibited by precipitation with ammonium sulphate, and the activity is lost after freezing.

AN AROMATIC AMINO ACID TRANSAMINASE FROM MUNG BEAN

A transaminase has been isolated and purified from young mung bean plants (Phaseolus aureus Roxb.). The enzyme catalyzes the transamination of phenylalanine in the presence of α-ketoglutarate with the production of equimolar amounts of phenylpyruvate and glutamate. Tyrosine and tryptophan also serve as substrates, and relative rate measurements indicate that only one enzyme is involved. In addition to α-ketoglutarate the enzyme also utilizes pyruvate, and to some extent glyoxylate and oxaloacetate as amino acceptors. The enzyme is stable in solution at 0–4 °C for several weeks, and acetone powders of the young plants stored at 0–4 °C retained their activity for several months. The enzyme is inhibited by precipitation with ammonium sulphate, and the activity is lost after freezing.

The production of hydrogen sulphide from thiosulphate by Escherichia coli.

Summary: Suspensions of non-proliferating Escherichia coli produced H2S from thiosulphate in the presence of pyruvate or acetaldehyde. Production of H2S was slight in the presence of α-ketoglutarate and α-ketobutyrate. Organic acids such as malate, fumarate, succinate, lactate, formate and acetate, aldehydes other than acetaldehyde and monohydric alcohols either had no effect or inhibited H2S production from thiosulphate. H2S was not produced from sulphite, bisulphite or sulphate, either in the presence or in the absence of the above-named compounds. Crude cell-free extracts of Escherichia coli produced H2S from thiosulphate in the presence of pyruvate. Experiments with dialysed extracts showed that inorganic phosphate, Mg ions and cocarboxylase were essential for H2S production. Treatment of the extracts with anion exchange resin revealed that in addition, coenzyme A was indispensable for H2S production from thiosulphate. The addition of DPN to extracts dialysed or treated with anion exchange resin did not influence H2S production to a marked degree.