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Results for “Succinate-CoA Ligases”

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At least 19 recordsLinked to original sources
PubMed1978

Affinity labeling of succinyl-CoA synthetase from porcine heart and Escherichia coli with oxidized coenzyme A disulfide.

Incubation of oxidized coenzyme A disulfide (produced by oxidation of reduced CoA with 1 eq of sodium periodiate or of CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA synthetase from either porcine heart or Escherichia coli led to the formation of inactive enzyme containing 1 mol of CoA per alphabeta dimer. The bound CoA was attached through a disulfide bond to a sulfhydryl group of the beta subunit. Release of CoA and restoration of activity was achieved by incubation of the modified enzyme with thiols, such as dithiothreitol. Interaction of oxidized CoA disulfide with enzyme was inhibited competitively by desulfo-CoA, which is a competitive inhibitor of the enzyme with respect to CoA. These data are evidence that oxidized CoA disulfide is an affinity label for the CoA binding site of succinyl-CoA synthetase and are the first positive results implicating the beta subunit in the catalytic mechanism of the enzyme.

Affinity Labels↗
PubMed1979

Menaquinone (vitamin K2) biosynthesis: conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid by Mycobacterium phlei enzymes.

The coenzyme A (CoA) and adenosine 5'-triphosphate-dependent conversion of o-succinylbenzoic acid (4-[2'-carboxyphenyl]-4-oxobutyric acid) to 1,4-dihydroxy-2-naphthoic acids is an important step in menaquinone (vitamin K2) biosynthesis. Cell-free extracts catalyzing this conversion, obtained from Mycobacterium phlei, were separated into three protein fractions by treatment with protamine sulfate. The second fraction (fraction B) and the supernatant (fraction S) alone did not catalyze dihydroxynaphthoate formation, but did so in combination. All of the results were consistent with the formation of an unstable intermediate, likely an o-succinylbenzoyl-CoA compound, by the action of fraction S. Adenosine 5'-triphosphate was required in this reaction and adenosine 5'-monophosphate was formed. This enzyme activity was termed o-succinylbenzoyl-CoA synthetase: the enzyme showed a marked stability to 0.1 N hydrochloric acid. The presumed o-succinylbenzoyl-CoA derivate was rather unstable; under a variety of conditions, it was converted to a spirodilactone form of o-succinylbenzoate. Fraction B contained an enzyme, termed naphthoate synthase, which converted the o-succinylbenzoyl-CoA derivative to 1,4-dihydroxy-2-naphthoate.

Adenosine Monophosphate↗
PubMed1978

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells.

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.

Cells, Cultured↗
PubMed1978

[Reaction mechanism of succinyl CoA synthetase from pigeon thoracic muscle].

The ability of succinyl-CoA-synthetase from pigeon thoracic muscle to interact with ATP is investigated. gamma-32P-ATP and 8-14C-ATP were used in experiments. It is found that the enzyme, when reacting with ATP in the presence of Mg2+, forms a complex containing 2 moles of ATP residue and 2 moles of phosphoric acid residue (splitted from ATP) per 1 mole of protein. After 2 hours of incubation at 0-4 degrees C, the complex is converted into another one, containing 4 residues of phosphoric acid per 1 mole of @protein. Both complexes are active, and their incubation with succinate and CoA results in the formation of succinyl-CoA. The reaction capacity of these enzyme complexes with some reaction substrates is investigated. The enzyme complex containing 2 phosphoric acid residues and 2 nucleotide residues is found to interact neither with CoA, nor with succinate. The enzyme complex containing 4 phosphoric acid residues does not react with CoA, but it interacts with 14C-succinate, releasing inorganic phosphate in the amount equivalent to the equimolar amount of protein-binding succinic acid.

Adenosine Triphosphate↗
PubMed1979

Evidence for a second histidine at the active site of succinyl-CoA synthetase from Escherichia coli.

Ethoxyformic anhydride was used to demonstrate the existence of a second important histidine in succinyl-CoA synthetase from Escherichia coli. Differential labeling of the enzyme by [3H]ethoxyformic anhydride gave a stoichiometry of one important histidine per alpha beta catalytic unit. Data are presented suggesting that this residue and an important thiol group on the beta subunit (Collier, G., and Nishimura, J.S. (1978) J. Biol. Chem. 253, 4938-4943) interact with each other during catalysis. A mechanism of action involving these 2 residues is proposed for one of the partial reactions catalyzed by succinyl-CoA synthetase.

Binding Sites↗
PubMed1977

Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons.

The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn. Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.

Adenosine Diphosphate↗
PubMed1975

Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs.

The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.

Bacteria↗