Publications

• Participation of Cys123alpha of Escherichia coli succinyl-CoA synthetase in catalysis.

  Hidber E., Brownie E.R., Hayakawa K., Fraser M.E.

  Succinyl-CoA synthetase has a highly conserved cysteine residue, Cys123alpha in the Escherichia coli enzyme, that is located near the CoA-binding site and the active-site histidine residue. To test whether the succinyl moiety of succinyl-CoA is transferred to the thiol of Cys123alpha as part of th ... >> More

  Succinyl-CoA synthetase has a highly conserved cysteine residue, Cys123alpha in the Escherichia coli enzyme, that is located near the CoA-binding site and the active-site histidine residue. To test whether the succinyl moiety of succinyl-CoA is transferred to the thiol of Cys123alpha as part of the catalytic mechanism, this residue was mutated to alanine, serine, threonine and valine. Each mutant protein was catalytically active, although less active than the wild type. This proved that the specific formation of a thioester bond with Cys123alpha is not part of the catalytic mechanism. To understand why the mutations affected catalysis, the crystal structures of the four mutant proteins were determined. The alanine mutant showed no structural changes yet had reduced activity, suggesting that the size of the cysteine is important for optimal activity. These results explain why this cysteine residue is conserved in the sequences of succinyl-CoA synthetases from different sources. << Less

  Acta Crystallogr. D 63:876-884(2007) [PubMed] [EuropePMC]

• Probing the nucleotide-binding site of Escherichia coli succinyl-CoA synthetase.

  Joyce M.A., Fraser M.E., Brownie E.R., James M.N., Bridger W.A., Wolodko W.T.

  Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purine nucleoside diphosphate, succinyl-CoA, and Pi with purine nucleoside triphosphate, succinate, and CoA via a phosphorylated histidine (H246alpha) intermediate. Two potential nucleotide-binding sites were predicted in the be ... >> More

  Succinyl-CoA synthetase (SCS) catalyzes the reversible interchange of purine nucleoside diphosphate, succinyl-CoA, and Pi with purine nucleoside triphosphate, succinate, and CoA via a phosphorylated histidine (H246alpha) intermediate. Two potential nucleotide-binding sites were predicted in the beta-subunit, and have been differentiated by photoaffinity labeling with 8-N3-ATP and by site-directed mutagenesis. It was demonstrated that 8-N3-ATP is a suitable analogue for probing the nucleotide-binding site of SCS. Two tryptic peptides from the N-terminal domain of the beta-subunit were labeled with 8-N3-ATP. These corresponded to residues 107-119beta and 121-146beta, two regions lying along one side of an ATP-grasp fold. A mutant protein with changes on the opposite side of the fold (G53betaV/R54betaE) was unable to be phosphorylated using ATP or GTP, but could be phosphorylated by succinyl-CoA and Pi. A mutant protein designed to probe nucleotide specificity (P20betaQ) had a Km(app) for GTP that was more than 5 times lower than that of wild-type SCS, whereas parameters for the other substrates remained unchanged. Mutations of residues in the C-terminal domain of the beta-subunit designed to distrupt one loop of the Rossmann fold (I322betaA, and R324betaN/D326betaA) had the greatest effect on the binding of succinate and CoA. They did not disrupt the phosphorylation of SCS with nucleotides. It was concluded that the nucleotide-binding site is located in the N-terminal domain of the beta-subunit. This implies that there are two active sites approximately 35 A apart, and that the H246alpha loop moves between them during catalysis. << Less

  Biochemistry 38:7273-7283(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural basis for the binding of succinate to succinyl-CoA synthetase.

  Huang J., Fraser M.E.

  Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To dete ... >> More

  Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate. << Less

  Acta Crystallogr. D 72:912-921(2016) [PubMed] [EuropePMC]

• Purification and properties of the phosphorylating enzyme from spinach.

  KAUFMAN S., ALIVISATOS S.G.

  J Biol Chem 216:141-152(1955) [PubMed] [EuropePMC]

• Studies on the mechanism of the reaction catalyzed by the phosphorylating enzyme.

  KAUFMAN S.

  J Biol Chem 216:153-164(1955) [PubMed] [EuropePMC]

• Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion.

  Elpeleg O., Miller C., Hershkovitz E., Bitner-Glindzicz M., Bondi-Rubinstein G., Rahman S., Pagnamenta A., Eshhar S., Saada A.

  The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-Co ... >> More

  The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the beta subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome. << Less

  Am. J. Hum. Genet. 76:1081-1086(2005) [PubMed] [EuropePMC]