Carboxylation Of propionyl-CoA Research Articles

Identification and metabolomic analysis of chemical elicitors for tacrolimus accumulation in Streptomyces tsukubaensis.

Tacrolimus is a widely used immunosuppressive agent in the treatment of various clinical diseases. However, the low fermentation yield seriously limits its further application. To stimulate tacrolimus synthesis, nine chemical elicitors of five groups were evaluated for their effects on tacrolimus accumulation in S. tsukubaensis. The results showed that sodium butyrate (SB), dimethylsulfoxide (DMSO), and LaCl3 could increase tacrolimus accumulation by more than 30%. Cumulative effects of different chemical elicitors exhibited that the highest tacrolimus yield was improved by 64.7% (303.60mg/L) in DMSO and La treatment, compared to the control. To decipher possible response mechanism, a weighted correlation network analysis (WGCNA) based on metabolomics was employed and datasets showed 13 distinct metabolic modules and 16 hub metabolites were possibly related to the stimulatory roles of DMSO, La, SB, and their combination treatments. The pathway analysis further exhibited that central carbon metabolism, amino acid metabolism, and fatty acid metabolism showed significant differences in the above chemical elicitor treatments. Thereinto, the carboxylation of propionyl-CoA from isoleucine and methionine degradation was first confirmed to be the main source of methylmalonyl-CoA by RT-PCR analysis in DMSO and La treatment. By further strengthening of the supply of methylmalonyl-CoA precursor in DMSO and La treatment, the final tacrolimus yield could reach to 372.12mg/L, 2.02-fold higher than the control. To our knowledge, this is the first study to unveil the potential mechanism of different chemical elicitor stresses in S. tsukubaensis based on metabolomics, and the established information provide valuable guidance for further improving tacrolimus production.

Propionyl-CoA carboxylase - A review.

Propionyl-CoA carboxylase (PCC) is the enzyme which catalyzes the carboxylation of propionyl-CoA to methylmalonyl-CoA and is encoded by the genes PCCA and PCCB to form a hetero-dodecamer. Dysfunction of PCC leads to the inherited metabolic disorder propionic acidemia, which can result in an affected individual presenting with metabolic acidosis, hyperammonemia, lethargy, vomiting and sometimes coma and death if not treated. Individuals with propionic acidemia also have a number of long term complications resulting from the dysfunction of the PCC enzyme. Here we present an overview of the current knowledge about the structure and function of PCC. We review an updated list of human variants which are published and provide an overview of the disease.

TetR Family Transcriptional Regulator PccD Negatively Controls Propionyl Coenzyme A Assimilation in Saccharopolyspora erythraea.

Propanol stimulates erythromycin biosynthesis by increasing the supply of propionyl coenzyme A (propionyl-CoA), a starter unit of erythromycin production in Saccharopolyspora erythraea Propionyl-CoA is assimilated via propionyl-CoA carboxylase to methylmalonyl-CoA, an extender unit of erythromycin. We found that the addition of n-propanol or propionate caused a 4- to 16-fold increase in the transcriptional levels of the SACE_3398-3400 locus encoding propionyl-CoA carboxylase, a key enzyme in propionate metabolism. The regulator PccD was proved to be directly involved in the transcription regulation of the SACE_3398-3400 locus by EMSA and DNase I footprint analysis. The transcriptional levels of SACE_3398-3400 were upregulated 15- to 37-fold in the pccD gene deletion strain (ΔpccD) and downregulated 3-fold in the pccD overexpression strain (WT/pIB-pccD), indicating that PccD was a negative transcriptional regulator of SACE_3398-3400. The ΔpccD strain has a higher growth rate than that of the wild-type strain (WT) on Evans medium with propionate as the sole carbon source, whereas the growth of the WT/pIB-pccD strain was repressed. As a possible metabolite of propionate metabolism, methylmalonic acid was identified as an effector molecule of PccD and repressed its regulatory activity. A higher level of erythromycin in the ΔpccD strain was observed compared with that in the wild-type strain. Our study reveals a regulatory mechanism in propionate metabolism and suggests new possibilities for designing metabolic engineering to increase erythromycin yield.IMPORTANCE Our work has identified the novel regulator PccD that controls the expression of the gene for propionyl-CoA carboxylase, a key enzyme in propionyl-CoA assimilation in S. erythraea PccD represses the generation of methylmalonyl-CoA through carboxylation of propionyl-CoA and reveals an effect on biosynthesis of erythromycin. This finding provides novel insight into propionyl-CoA assimilation, and extends our understanding of the regulatory mechanisms underlying the biosynthesis of erythromycin.

Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C4-dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica, succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii, and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as characteristic enzymes of this cycle.

Triheptanoin partially restores levels of tricarboxylic acid cycle intermediates in the mouse pilocarpine model of epilepsy

Triheptanoin, the triglyceride of heptanoate, is anticonvulsant in various epilepsy models. It is thought to improve energy metabolism in the epileptic brain by re-filling the tricarboxylic acid (TCA) cycle with C4-intermediates (anaplerosis). Here, we injected mice with [1,2-(13) C]glucose 3.5-4 weeks after pilocarpine-induced status epilepticus (SE) fed either a control or triheptanoin diet. Amounts of metabolites and incorporations of (13) C were determined in extracts of cerebral cortices and hippocampal formation and enzyme activity and mRNA expression were quantified. The percentage enrichment with two (13) C atoms in malate, citrate, succinate, and GABA was reduced in hippocampal formation of control-fed SE compared with control mice. Except for succinate, these reductions were not found in triheptanoin-fed SE mice, indicating that triheptanoin prevented a decrease of TCA cycle capacity. Compared to those on control diet, triheptanoin-fed SE mice showed few changes in most other metabolite levels and their (13) C labeling. Reduced pyruvate carboxylase mRNA and enzyme activity in forebrains and decreased [2,3-(13) C]aspartate amounts in cortex suggest a pyruvate carboxylation independent source of C-4 TCA cycle intermediates. Most likely anaplerosis was kept unchanged by carboxylation of propionyl-CoA derived from heptanoate. Further studies are proposed to fully understand triheptanoin's effects on neuroglial metabolism and interaction.

N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition

Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP-binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS.

Molecular analysis of PCCB gene in Korean patients with propionic acidemia

Propionic acidemia (PA) is an autosomal recessive inborn error in the catabolism of methionine, isoleucine, threonine, and valine, odd-numbered chain length fatty acids and cholesterol. Clinical symptoms are very heterogeneous and present as a severe neonatal-onset or a late-onset form. It is caused by a deficiency of propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a biotin-dependent enzyme that catalyzes the carboxylation of propionyl-CoA to d-methylmalonyl-CoA. PCC is a heteropolymeric enzyme composed of α- and β-subunits. A greater heterogeneity is observed in the PCCA gene, while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. We identified eight Korean patients with PA by organic acid analysis confirmed in five patients by the PCC enzyme assay in the lymphoblasts. Two neonatal-onset patients showed undetectable PCC activities while three cases with residual enzyme activities had relatively late manifestations. In the molecular analysis, we identified five novel mutations, Y439C, 1527del3, 1357insT, IVS12–8T → A, and 31del10, and one known mutation, T428I in PCCB gene. Alleleic frequency of T428I in Korean patients with PA was 56.3% in this study. Two neonatal-onset patients with null enzyme activities were homozygotes with 1527del3 and T428I, respectively. This finding implies that T428I and 1527del3 mutation could be responsible for their severe clinical courses and null enzyme activities. The mRNA of PCCB gene in T428I and 1527del3 homozygotes were normal but in Western blot analysis, the βPCC-subunit was only absent in 1527del3 homozygote patient suggesting different molecular pathology.

Structure of the PCCA Gene and Distribution of Mutations Causing Propionic Acidemia

Propionyl-CoA carboxylase (PCC, EC 6.4.1.3) is a mitochondrial, biotin-dependent enzyme that functions in the catabolism of branched-chain amino acids, fatty acids with odd-numbered chain lengths, and other metabolites. It catalyzes the ATP-dependent carboxylation of propionyl-CoA to d-methylmalonyl-CoA. PCC is composed of two types of subunits, likely as α4β4 or α6β6, with the α subunit containing the covalently bound biotin prosthetic group. A genetic deficiency of PCC activity causes propionic acidemia, a potentially fatal disease with onset in severe cases in the newborn period. Affected patients may have mutations of either the PCCA or PCCB gene. In this study, we have determined the structure of the human PCCA gene which, at the present time, is only partially represented in the databases. Based on reported ESTs and confirmed by RT-PCR, we also redefine the translation initiation codon to a position 75 nucleotides upstream of the currently accepted initiation codon. We show the distribution of mutations, including three identified in this study, and renumber all reported mutations to count from the new initiation codon. The gene spans more than 360 kb and consists of 24 exons ranging from 37 to 335 bp in length. The introns range in size from 104·bp to 66 kb. We have also determined the nucleotide sequence of ∼1 kb of the 5′-flanking region upstream of the ATG translation initiation site. The proximal 400 bp of the 5′-flanking region shows a high G + C content (67%) and is part of a putative 1-kb CpG island that extends into exon 1 and part of intron 1. The putative promoter lacks a TATA box but contains two AP-1 sites and a conservatively defined consensus GC box, the latter characteristic of the core binding sequence of the Sp1 transcription factor.

Changes in the Carboxyl Terminus of the β Subunit of Human Propionyl-CoA Carboxylase Affect the Oligomer Assembly and Catalysis: Expression and Characterization of Seven Patient-Derived Mutant Forms of PCC in Escherichia coli

Propionyl-CoA carboxylase (PCC) catalyzes the biotin-dependent carboxylation of propionyl-CoA to d-methylmalonyl-CoA in the mitochondrial matrix. Human PCC is a dodecamer composed of pairs of nonidentical α and β subunits encoded by PCCA and PCCB genes, respectively. Deficiency of PCC results in propionic acidemia (PA), a metabolic disorder characterized by severe metabolic ketoacidosis, vomiting, lethargy, and hypotonia. To date, almost 60 mutations have been reported in both genes. Exon 15 of the β subunit is one of the two sites where a number of mutations have been identified in PA patients. In the primary βPCC sequence, these mutations lead to three substitutions (R512C, L519P, and N536D), three truncations (R499X, R514X, and W531X), and one insertion (A51_R514insP). We expressed these mutant proteins in Escherichia coli in which the GroESL complex was overexpressed. The only mutation that does not impact the stability of mutant βPCC in bacteria is W531X. The remaining mutations lead to either complete (L519P, N536D) or partial (R499X, R512C, A513_R514insP, and R514X) degradation of the mutant subunits. Size-exclusion chromatography revealed that R512C and W531X do not affect the assembly of αPCC and βPCC to active oligomers. Specific activities for these mutant proteins, however, were only 3.9 and 10% of the wild type, respectively. Taken together, the carboxyl-terminal portion of 40 amino acid residues of the β subunit affects the stability and the assembly of the α and β subunits as well as the carboxylation of propionyl-CoA.

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.

Studies on the Plant Methylcrotonyl-CoA Carboxylase 1

Summary In our research on the presence of biotin-containing carboxylases in plants we found, in addition to acetyl-CoA carboxylase (ACC), the carboxylation of propionyl-CoA (PCC-activity) and methylcrotonylCoA (MCC-activity) in protein preparations from maize, oat, barley, pea and lentil. Whether the PCCactivity originates from a distinct, separate enzyme or represents a side-activity of the ACC is not yet clear. The carboxylation of methylcrotonyl-CoA is clearly catalyzed by a new biotin enzyme, the methylcrotonyl-CoA carboxylase (MCC), which can be inhibited by avidin. It is not present in isolated chloroplasts, but seems to be a component of plant mitochondria. In contrast to ACC and PCC, the MCC of barley and maize is not inhibited by two herbicide groups, known to be strong inhibitors of the ACC from grasses. The activity of ACC and MCC from maize exhibit a quite different developmental pattern: ACC is found in young leaf tissue, whereas MCC shows up only in older tissue. The MCC from barley was purified 60-fold using different HPLC columns.

Biosynthesis of the polyether antibiotic monensin-A. Results from the incorporations of labelled acetate and propionate as a probe of the carbon chain assembly processes

The incorporation of sodium [2-2H2]- and (S)-[2-2H1]-propionate into the polyether antibiotic monensin-A in cultures of Streptomyces cinnamonensis occurs with retention of label only at C-4 and C-6, whereas during the incorporation of sodium (R)-[2-2H1]propionate the deuterium label is lost to the medium. These results are consistent with the formation of (S)-methylmalonyl-CoA from the labelled propionate by carboxylation of propionyl-CoA with loss of the 2-pro-R hydrogen. The (S)-methylmalonyl-CoA is subsequently incorporated into the antibiotic by a decarboxylative condensation occurring with overall inversion. The incorporations of sodium [1-13C,2-2-H3]-and [2-13C,2-2H3]-acetates into monensin-A provide evidence for a pathway of metabolism leading to methylmalonyl-CoA that does not proceed via succinyl-CoA. Instead, acetyl-CoA may be processed via butyryl-CoA and isobutyryl-CoA, to afford (S)-methylmalonyl-CoA.

Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus

Streptomyces erythreus produces erythromycin presumably from methylmalonyl-coenzyme A, (CoA) which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA, and butyryl-CoA was purified to near homogeneity from S. erythreus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose, and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a K m of 0.09 m m and V of 0.86μmol/mg/min, acetyl-CoA (16%) with a K m of 0.17 m m and V of 0.08μmol/mg/min, and butyryl-CoA (7.7%) with a K m of 0.67 m m and V of 0.044 μmol/mg/min. The native enzyme has a molecular weight of 537,000 and consists of two types of subunits with molecular weights of 67,000 and 61,000, respectively, indicating an octameric α 4 β 4 type of structure. Biotin is associated with the large subunit (α). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (three- to fourfold) by K +, Rb + and Cs + but not by Na + or Li + and is inhibited by high concentrations of NH 4 + and C1 −. Neither citrate nor free CoA stimulated the enzyme. The enzyme was shown to be stereospecific and generated only S-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the first enzyme possibly involved in erythromycin production to be isolated in homogeneous form.

Metabolic effects of pent-4-enoate in isolated perfused rat heart.

The metabolic effects of the hypoglycaemic agent pent-4-enoate were studied in isolated, beating or potassium-arrested rat hearts. The addition of 0.8mM-pent-4-enoate to the perfusion fluid increased O2 consumption by 76% in the arrested heart and by 14% in the beating heart; the concentration ratio of phosphocreatine/creatine increase concomitantly by 47% and 27% respectively. Perfusion of the heart with pent-4-enoate resulted in a 30-fold increase in the concentration of the pool of tricarboxylic acid-cycle intermediates in the tissue, about 90% of this increase being due to malate. The sum of the concentrations of the myocardial free amino acids remained virtually unchanged during the accumulation of the tricarboxylic acid-cycle intermediates. It was concluded that pent-4-enoate can be effectively metabolized in the myocardium and that its metabolism probably proceeds via propionyl-CoA, since pent-4-enoate reproduces many of the metabolic characteristics of propionate in the cardiac muscle. The accumulation of the tricarboxylic acid-cycle intermediates is probably due to carboxylation of propionyl-CoA. The response pattern of the metabolite concentrations in the cardiac muscle is quite different from that in the liver, in which decrease of the concentrations of the tricarboxylic acid-cycle intermediates has been observed previously [Williamson, Rostand & Peterson (1970) J. Biol. Chem. 245, 3242-3251].

Acute Neonatal Nonketotic Hyperglycinemia: Normal Propionate and Methylmalonate Metabolism

Propionyl-CoA carboxylase and combined methylmalonyl-CoA (MMA-CoA) racemase and -mutase activities were studied in liver and fibroblasts of two patients with the acute neonatal form of nonketotic hyperglycemia. In all experiments, these enzyme activities studied in tissues of the patients were within the range of healthy control subjects, whereas no propionyl-CoA carboxylase activity was measurable in the fibroblasts of a patient with propionic acidemia. Subcellular fractionation of liver and fibroblasts indicated that the normal amounts of MMA-CoA found after incubation of whole tissue homogenate were formed by propionyl-CoA carboxylase, a mitochondrial enzyme, and not be acetyl-CoA carboxylase, which theoretically could also be involved in the carboxylation of propionyl-CoA. From the above data as well as from clinical and biochemical observations in three patients, it was concluded that there exists a true nonketotic hyperglycinemia which is not related etiologically to the different disorders of the ketotic hyperglycinemia syndrome. True nonketotic hyperglycinemia is not associated with ketoacidosis even after loading with propionate- and MMA precursors. It must be distinguished by exclusion from mild forms of the ketotic hyperglycinemia syndrome which may present clinically as hyperglycinemia without ketosis.

Lipid biosynthesis in sebaceous glands. Regulation of the synthesis of n- and branched fatty acids by malonyl-coenzyme A decarboxylase

Crude cell-free extracts isolated from the uropygial glands of goose catalyzed the carboxylation of propionyl-CoA but not acetyl-CoA. However, a partially purified preparation catalyzed the carboxylation of both substrates and the characteristics of this carboxylase were similar to those reported for chicken liver carboxylase. The Km and Vmax for the carboxylation of either acetyl-CoA or propionyl-CoA were 1.5 times 10- minus-5 M and 0.8 mumol per min per mg, respectively. In the crude extracts an inhibitor of the acetyl-CoA carboxylase activity was detected. The inhibitor was partially purified and identified as a protein that catalyzed the rapid decarboxylation of malonyl-CoA. This enzyme was avidin-insenitive and highly specific for malonyl-CoA with very low rates of decarboxylation for methylmalonyl-CoA and malonic acid. Vmax and Km for malonyl-CoA decarboxylation, at the pH optimum of 9.5, were 12.5 mumol per min per mg and 8 times 10- minus-4 M, respectively. The relative activities of the acetyl-CoA carboxylase and malonyl-CoA decarboxylase were about 4 mumol per min per gland and 70 mumoles per min per gland, respectively. Therefore acetyl-CoA and methylmalonyl-CoA should be the major primer and elongating agent, respectively, present in the gland. The major fatty acid formed from these precursors by the fatty acid synthetase of the gland would be 2,4,6,8-tetramethyl-decanoic acid which is known to be the major fatty acid of the gland (Buckner, J. S. and Kolattukudy, P. E. (1975), Biochemistry, following paper). Therefore it is concluded that the malonyl-CoA decarboxylase controls fatty acid synthesis in this gland.

Acceleration of gluconeogenesis from propionate by Dl-carnitine in the rat kidney cortex.

1. The rate of gluconeogenesis from propionate in rat kidney-cortex slices was stimulated up to 3.5-fold by dl-carnitine and by bicarbonate, and was inhibited by inorganic phosphate or high concentrations of propionate (above 3mm). 2. The stimulatory effect of carnitine was dependent on the bicarbonate concentration and could be replaced at low propionate concentration by addition of 25mm-bicarbonate-carbon dioxide buffer. At low bicarbonate concentration the carnitine concentration can be rate-limiting. 3. All observations are in accordance with the view that the action of carnitine is in principle the same as that established for other fatty acids in other tissues, namely that carnitine promotes the appearance of propionyl-CoA within the mitochondrion by acting as a carrier. 4. The accelerating effects of carnitine and bicarbonate and the inhibitory effect of phosphate can be explained on the basis of the known properties of key enzymes of propionate metabolism, i.e. the reversibility of the reactions leading to the formation of methylmalonyl-CoA from propionyl-CoA. 5. 5mm-Propionate caused a five- to ten-fold fall in the free CoA content of the tissue. This fall can account for the inhibition of respiration and gluconeogenesis caused by high propionate concentration. 6. Relatively large quantities of propionyl-l-carnitine (15% of the propionate removed) were formed when dl-carnitine was present; thus the ;activation' of propionate proceeded at a faster rate than the carboxylation of propionyl-CoA. The metabolism of added propionyl-l-carnitine was accompanied by glucose synthesis. 7. The appearance of radioactivity from [2-(14)C]propionate in both glucose and carbon dioxide was as expected on account of the randomization of C-2 and C-3 of propionate, i.e. the formation of succinate as an intermediate. 8. The maximum rate of glucose synthesis from propionate (93.3+/-3.3mumoles/g. dry wt./hr.) was not affected by dietary changes aimed at varying the rate of caecal volatile fatty acid formation in the rat. 9. Inhibition of gluconeogenesis by high propionate concentration was not found in those species where the rate of caecal or ruminal propionate production is high under normal conditions (rabbit, sheep and cow).

On the oxidation of α-oxobutyrate by isolated mammalian mitochondria

The oxidation of α-oxobutyrate in rat kidney and liver mitochondria depleted of endogenous ATP has been studied. The use of malonate and atractyloside as inhibitors made it possible to identify the compartmentation of the successive steps in the mitochondrial structure. It is suggested that oxidative decarboxylation of α-oxobutyrate occurs only inside the atractyloside-sensitive barrier ( i.e. the inner membrane), while the carboxylation step i.e. the carboxylation of propionyl-CoA to methylmalonyl-CoA may occur either inside or outside this barrier.

The Enzymatic Carboxylation of Propionyl Coenzyme A: STUDIES INVOLVING DEUTERATED AND TRITIATED SUBSTRATES

Abstract (R)- and (S)-2-deuteriopropionyl coenzyme A of approximately 75% optical purity were prepared by a series of reactions starting from optically pure alanine. With these compounds as substrates for propionyl-CoA carboxylase, the deuterium isotope effect was found to be small and probably secondary. No tritium isotope effect was observed when enzymatically prepared 2-3H-propionyl-CoA was used as substrate. The rate of release of tritium was the same as that of the over-all reaction. Tritium from 3H2O was not incorporated into propionyl-CoA in the absence of the reverse reaction. The data were consistent with a concerted mechanism in which an α-proton was removed simultaneously with carboxylation of propionyl-CoA. By various techniques the reaction was shown to proceed with retention of configuration about the α-carbon.

Further studies on the mechanism of the enzymic carboxylation of propionyl-CoA

Further studies on the mechanism of the enzymic carboxylation of propionyl-CoA