Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.
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ABSTRACT:
SUBMITTER: Sharp JA
PROVIDER: S-EPMC1152106 | biostudies-other | 1983 Jul
REPOSITORIES: biostudies-other
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Project description:CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide 'mapping' and N-terminal analysis.
| S-EPMC1185841 | biostudies-other
Project description:The amounts of succinyl-CoA--3-oxo acid CoA-transferase (EC 2.8.3.5) decrease progressively in skeletal muscle in streptozotocin-diabetic rats, reaching after 10 days about 50% of the value in normal rat muscle. Electrofocusing studies indicate the occurrence of partial proteolysis of the enzyme in diabetic muscle. However, several functional parameters relating to acetoacetate utilization, including substrate inhibition, are quite similar for muscle transferase preparations from normal and diseased rats. The development of pathological ketoacidosis is discussed in the light of these observations.
| S-EPMC1163999 | biostudies-other
Project description:1. Tissue activities, intracellular distribution as well as selected kinetic and molecular properties of succinyl-CoA-3-oxo acid CoA transferase (EC 2.8.3.5), which is an initiator of ketone body usage, were examined in rat kidney, heart, brain, skeletal muscle and liver. 2. The activities of the transferase in these tissues are similar to reported values and are somewhat affected by the homogenization medium. Higher recoveries of activity are obtained when a phosphate buffer is used during the homogenization; Tris solutions containing sucrose and mannitol lead to only slightly lower recoveries, but can be used in studies to determine the subcellular localization of the transferase activity. 3. A close correlation was observed between the relative activities of citrate synthase (a mitochondrial marker enzyme) and CoA transferase in the cytoplasmic, particulate and mitochondrial fractions from the five tissues. 4. The K(m) values for acetoacetate (measured in two different ways), the ratio of V(max.) values for the two enzyme-catalysed half-reactions, and succinate product inhibition are quite similar for the enzyme from each tissue. 5. The enzymes are also similar in molecular weight (with an approx. mol.wt. of 100000 as determined by gel filtration). All show an active band in isoelectric-focusing studies with pI 7.6, except for the enzyme from heart (pI 6.8). 6. The results demonstrate a mitochondrial origin for CoA transferase in these rat tissues and support the proposition that CoA transferase is a ketolytic enzyme, i.e. an enzyme uniquely involved in the complete oxidation of ketone bodies. The structural and functional similarities of these transferases suggest that factors other than differences in K(m) values account for differences in the utilization of ketone bodies by various tissues.
| S-EPMC1168327 | biostudies-other
Project description:1. The maximum activities of hexokinase and phosphofructokinase in nervous tissue from 18 different animals from different phyla range from 5.1 to 17.6 and from 24.0mumol/min per g fresh wt. respectively. In any one tissue the activities of these two enzymes are, in general, very similar. The rate of glucose utilization by the brain in vivo is much lower than the activities of hexokinase or phosphofructokinase. It is suggested that the high activities of these enzymes indicate a capacity for glycolysis which may be used by the brain during hypoxia or during conditions of extreme neuronal activity. 2. The activities of 3-oxo acid CoA-transferase and acetoacetyl-CoA thiolase in the nervous tissues range from 1.1 to 15.3 and from 0.7 to 4.5mumol/min per g fresh wt. respectively. Unfortunately the activities of these enzymes cannot be used to estimate maximal flux through the ketone-body-utilization pathway, since they may catalyse reactions that are close to equilibrium. Nonetheless, the presence of these enzymes in nervous tissue from a large variety of animals suggests that the importance of ketone bodies as a fuel for nervous tissue may be widespread in the animal kingdom.
| S-EPMC1177791 | biostudies-other
Project description:Mouse glutathione S-transferase GST YfYf (an orthologue of GST P or 7-7 in the rat and of GST pi in the human) was found to have a subunit Mr of 24,500 and cross-reacted with anti-(rat GST YfYf). N-Terminal analysis showed a close similarity to the rat, human and bovine orthologues. On isoelectric focusing the native enzyme had a pI of 8.3 and a pI of 7.3 in the presence of urea. Initial-rate studies with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates and inhibition studies with the product of the enzyme-catalysed conjugation of CDNB and GSH, S-(2,4-dinitrophenyl)glutathione, indicated a rapid-equilibrium random mechanism for the enzyme. The diuretic drug ethacrynic acid was found to be simultaneously a competitive inhibitor and an uncompetitive activator of the enzyme (with CDNB as the substrate whose concentration was varied). By using a computer simulation program (EKPLOT) a model was developed that would explain the experimental data. It is proposed that ethacrynic acid can compete with CDNB at the active site but simultaneously bind to an allosteric site on the enzyme, causing an elevation in the Vmax. for the conjugation of CDNB and GSH. The implications of such an activation mechanism for an enzyme potentially conjugating a range of xenobiotic compounds are discussed.
| S-EPMC1150111 | biostudies-other
Project description:Microbes tailor macromolecules and metabolism to overcome specific environmental challenges. Acetic acid bacteria perform the aerobic oxidation of ethanol to acetic acid and are generally resistant to high levels of these two membrane-permeable poisons. The citric acid cycle (CAC) is linked to acetic acid resistance in Acetobacter aceti by several observations, among them the oxidation of acetate to CO2 by highly resistant acetic acid bacteria and the previously unexplained role of A. aceti citrate synthase (AarA) in acetic acid resistance at a low pH. Here we assign specific biochemical roles to the other components of the A. aceti strain 1023 aarABC region. AarC is succinyl-coenzyme A (CoA):acetate CoA-transferase, which replaces succinyl-CoA synthetase in a variant CAC. This new bypass appears to reduce metabolic demand for free CoA, reliance upon nucleotide pools, and the likely effect of variable cytoplasmic pH upon CAC flux. The putative aarB gene is reassigned to SixA, a known activator of CAC flux. Carbon overflow pathways are triggered in many bacteria during metabolic limitation, which typically leads to the production and diffusive loss of acetate. Since acetate overflow is not feasible for A. aceti, a CO(2) loss strategy that allows acetic acid removal without substrate-level (de)phosphorylation may instead be employed. All three aar genes, therefore, support flux through a complete but unorthodox CAC that is needed to lower cytoplasmic acetate levels.
| S-EPMC2447011 | biostudies-literature
Project description:The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbon–limited chemostat with phenylalanine as nitrogen source indicated that: i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate α-keto acid decarboxylase. ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any α-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway. Keywords: Strain comparison
2007-11-15 | GSE9590 | GEO
Project description:Coenzyme A (CoA) transferases catalyze reversible transfer of CoA groups from CoA-thioesters to free acids, playing important roles in the metabolism of carboxylic acids in all organisms. An intramolecular CoA transferase, Mesaconyl-CoA C1-C4 CoA transferase (MCT) was identified in the autotrophic CO2 fixation pathway, 3-hydroxypropionic acid cycle of filamentous anoxygenic phototrophs (FAPs). Different from the well-known CoA transferases that catalyze CoA transfer between two distinct substrates, MCT specifically catalyzes the reversible transformation of mesaconyl-C1-CoA to mesaconyl-C4-CoA, a key reaction intermediate for carbon fixation. However, the molecular mechanism of MCT in employing one substrate is enigmatic. Here we determined the crystal structure of MCT from a chlorosome-less FAP Roseiflexus castenholzii at 2.5 Å resolution, and characterized the catalytic mechanisms through structural analyses and molecular dynamic simulations. The structure of R. castenholzii MCT consists of a Rossmann fold larger domain and a small domain that are connected by two linkers. Two MCT subunits are cross interlocked at the linker regions to form a functional dimer in solution, in which the substrate binding pockets are located at the interface of the Rossmann fold larger domain from one subunit and the small domain from the other subunit. In the simulated binding structures, both the substrate mesaconyl-C1-CoA and product mesaconyl-C4-CoA form extensive electrostatic and hydrogen bonding interactions with MCT. But some differences exist in the binding mode of these two CoA analogs, Arg314' from the second subunit of the dimer presenting dramatic conformational changes in binding with mesaconyl-C4-CoA. Together with Arg47 and one water molecule, a strictly conserved residue Asp165 are essential for catalyzing the reversible intramolecular CoA transfer reaction, through the electrostatic and hydrogen bonding interactions with the mesaconic tail of both the substrate and product. This study revealed a previously unrecognized mechanism for the uncommon intramolecular CoA transfer reaction, which will not only broaden the knowledge on the catalytic mechanisms of CoA transferases, but also contribute to enzyme engineering or biosynthetic applications of the 3-HP cycle for synthesis of fine chemicals and important metabolites.
| S-EPMC9196870 | biostudies-literature
Project description:The synthesis of polymerizable 7-(methacroyloxy)-2-oxo-heptylphosphonic acid M1 destined for self-etch adhesives is described. M1 is characterized by 1H, 13C and 31P-NMR spectroscopy. Its homopolymerization and copolymerization reactivity in the solvents methanol and dioxane between 45 and 70°C in the presence of azobisisobutyronitrile (AIBN) are examined. Polymerization proceeds readily through a thermal free radical initiation. The intensity exponents for the monomer and initiator are only slightly over 1 and approximately 0.5, respectively. This is in accordance with the results typically observed for an ideal free radical polymerization with termination mainly by disproportionation, which is typical for methyl methacrylate (MMA) homopolymerization. The kinetics of copolymerization with MMA are monitored by online 1H-NMR spectroscopy. Two copolymerization reactions for each pair of co-monomers are sufficient to evaluate the copolymerization parameters using the Jaacks method, the Fineman-Ross method and the nonlinear least-squares method. All three methods give similar results for particular monomer M1/MMA couple.
| S-EPMC6419686 | biostudies-literature
Project description:Bacterial formyl-CoA:oxalate CoA-transferase (FCOCT) and oxalyl-CoA decarboxylase work in tandem to perform a proton-consuming decarboxylation that has been suggested to have a role in generalized acid resistance. FCOCT is the product of uctB in the acidophilic acetic acid bacterium Acetobacter aceti. As expected for an acid-resistance factor, UctB remains folded at the low pH values encountered in the A. aceti cytoplasm. A comparison of crystal structures of FCOCTs and related proteins revealed few features in UctB that would distinguish it from nonacidophilic proteins and thereby account for its acid stability properties, other than a strikingly featureless electrostatic surface. The apparently neutral surface is a result of a "speckled" charge decoration, in which charged surface residues are surrounded by compensating charges but do not form salt bridges. A quantitative comparison among orthologs identified a pattern of residue substitution in UctB that may be a consequence of selection for protein stability by constant exposure to acetic acid. We suggest that this surface charge pattern, which is a distinctive feature of A. aceti proteins, creates a stabilizing electrostatic network without stiffening the protein or compromising protein-solvent interactions.
| S-EPMC3403466 | biostudies-literature