Publications

• Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA.

  Fraser M.E., Hayakawa K., Brown W.D.

  Catalysis by succinyl-CoA:3-oxoacid CoA transferase proceeds through a thioester intermediate in which CoA is covalently linked to the enzyme. To determine the conformation of the thioester intermediate, crystals of the pig enzyme were grown in the presence of the substrate acetoacetyl-CoA. X-ray ... >> More

  Catalysis by succinyl-CoA:3-oxoacid CoA transferase proceeds through a thioester intermediate in which CoA is covalently linked to the enzyme. To determine the conformation of the thioester intermediate, crystals of the pig enzyme were grown in the presence of the substrate acetoacetyl-CoA. X-ray diffraction data show the enzyme in both the free form and covalently bound to CoA via Glu305. In the complex, the protein adopts a conformation in which residues 267-275, 280-287, 357-373, and 398-477 have shifted toward Glu305, closing the enzyme around the thioester. Enzymes provide catalysis by stabilizing the transition state relative to complexes with substrates or products. In this case, the conformational change allows the enzyme to interact with parts of CoA distant from the reactive thiol while the thiol is covalently linked to the enzyme. The enzyme forms stabilizing interactions with both the nucleotide and pantoic acid portions of CoA, while the interactions with the amide groups of the pantetheine portion are poor. The results shed light on how the enzyme uses the binding energy for groups remote from the active center of CoA to destabilize atoms closer to the active center, leading to acceleration of the reaction by the enzyme. << Less

  Biochemistry 49:10319-10328(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

  Corthesy-Theulaz I.E., Bergonzelli G.E., Henry H., Bachmann D., Schorderet D.F., Blum A.L., Ornston L.N.

  Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori s ... >> More

  Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori strains tested by polymerase chain reaction or slot blotting but not in Campylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed in Escherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in different H. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits of Mr approximately 26,000 and 24, 000 that are both necessary for H. pylori CoA-transferase function. The lack of alpha-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism. << Less

  J. Biol. Chem. 272:25659-25667(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of the mammalian CoA transferase from pig heart.

  Bateman K.S., Brownie E.R., Wolodko W.T., Fraser M.E.

  Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based o ... >> More

  Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based on the three-dimensional structure of a CoA transferase determined from an anaerobic bacterium that utilizes its enzyme for glutamate fermentation. To better interpret clinical data, we have determined the structure of a mammalian CoA transferase from pig heart by X-ray crystallography to 2.5 A resolution. Instrumental to the structure determination were selenomethionine substitution and the use of argon during purification and crystallization. Although pig heart SCOT adopts an alpha/beta protein fold, resembling the overall fold of the bacterial CoA transferase, several loops near the active site of pig heart SCOT follow different paths than the corresponding loops in the bacterial enzyme, accounting for differences in substrate specificities. Two missense mutations found associated with SCOT of ketoacidosis patients were mapped to a location in the structure that might disrupt the stabilization of the amino-terminal strand and thereby interfere with the proper folding of the protein into a functional enzyme. << Less

  Biochemistry 41:14455-14462(2002) [PubMed] [EuropePMC]

• Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A.

  Tammam S.D., Rochet J.C., Fraser M.E.

  Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), refl ... >> More

  Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28 or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys 28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein was unexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a different conformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On the basis of these results and the studies of the structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17 degrees coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate or acetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has led to the proposal of a new mechanism for catalysis by SCOT. << Less

  Biochemistry 46:10852-10863(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of succinyl-CoA:3-oxo-acid CoA-transferase from rat brain.

  Russell J.J., Patel M.S.

  Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weigh ... >> More

  Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by G-150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 mumol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate leads to acetoacetyl-CoA) are consistent with a "ping pong" mechanism. Substrate inhibition appears above approximately 1 mM acetoacetate. Apparent Km values were 70 microM for acetoacetate and 156 microM for succinyl-CoA (the forward reaction), and 59 microM for acetoacetyl-CoA and 25 mM for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart. << Less

  J Neurochem 38:1446-1452(1982) [PubMed] [EuropePMC]

• Pig heart CoA transferase exists as two oligomeric forms separated by a large kinetic barrier.

  Rochet J.C., Brownie E.R., Oikawa K., Hicks L.D., Fraser M.E., James M.N., Kay C.M., Bridger W.A., Wolodko W.T.

  Pig heart CoA transferase (EC 2.8.3.5) has been shown previously to adopt a homodimeric structure, in which each subunit has a molecular weight of 52 197 and consists of N- and C-domains linked by a hydrophilic linker or "hinge". Here we identify and characterize a second oligomeric form constitue ... >> More

  Pig heart CoA transferase (EC 2.8.3.5) has been shown previously to adopt a homodimeric structure, in which each subunit has a molecular weight of 52 197 and consists of N- and C-domains linked by a hydrophilic linker or "hinge". Here we identify and characterize a second oligomeric form constituent in purified enzyme preparations, albeit at low concentrations. Both species catalyze the transfer of CoA with similar values for k(cat) and K(M). This second form sediments more rapidly than the homodimer under the conditions of conventional sedimentation velocity and active enzyme centrifugation. Apparent molecular weight values determined by sedimentation equilibrium and gel filtration chromatography are 4-fold greater than the subunit molecular weight, confirming that this form is a homotetramer. The subunits of both oligomeric forms are indistinguishable with respect to molecular mass, far-UV CD, intrinsic tryptophan fluorescence, and equilibrium unfolding. Dissociation of the homotetramer to the homodimer occurs very slowly in benign solutions containing high salt concentrations (0.25-2.0 M KCl). The homotetramer is fully converted to homodimer during refolding from denaturant at low protein concentrations. Disruption of the hydrophilic linker between the N- and C-domains by mutagenesis or mild proteolysis causes a decrease in the relative amount of the larger conformer. The homotetramer is stabilized by interactions involving the helical hinge region, and a substantial kinetic barrier hinders interconversion of the two oligomeric species under nondenaturing conditions. << Less

  Biochemistry 39:11291-11302(2000) [PubMed] [EuropePMC]

• Dimeric pig heart succinate-coenzyme A transferase uses only one subunit to support catalysis.

  Lloyd A.J., Shoolingin-Jordan P.M.

  Pig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate ... >> More

  Pig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate in which the active site glutamate (E344) of both subunits each forms thiolester links with coenzyme A. Reaction of this dimeric enzyme-coenzyme A species with sodium borohydride leads to inactivation of the enzyme and reduction of the thiolester on both subunits to the corresponding enzyme alcohol, as judged by electrospray mass spectrometry. Reaction of the dimeric enzyme-coenzyme A intermediate with either succinate or acetoacetate, however, results in only one-half of the coenzyme A being transferred to the acceptor carboxylate to form either succinyl-coenzyme A or acetoacetyl-coenzyme A. Reaction of this latter enzyme species with borohydride caused no loss of enzyme activity despite the reduction of the remaining half of the enzyme-coenzyme A thiolester to the enzyme alcohol. That this catalytic asymmetry existed between subunits within the same enzyme dimer was demonstrated by showing that the enzyme species, created by successive reaction with acetoacetyl-coenzyme A and succinate, bound to Blue MX-2G Sepharose through the remaining available active site and could be eluted as a single chromatographic species by succinyl-coenzyme A. It is concluded that while both of the subunits of the succinate-coenzyme A transferase dimer are able to form enzyme-coenzyme A thiolester intermediates, only one subunit is competent to transfer the coenzyme A moiety to a carboxylic acid acceptor to form the new acyl-coenzyme A product. The possible structural basis for this catalytic asymmetry and its mechanistic implications are discussed. << Less

  Biochemistry 40:2455-2467(2001) [PubMed] [EuropePMC]

• Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.

  Sharp J.A., Edwards M.R.

  The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of ... >> More

  The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed. << Less

  Biochem J 213:179-185(1983) [PubMed] [EuropePMC]

• Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase.

  Rochet J.C., Bridger W.A.

  The enzyme CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase [3-oxoacid CoA transferase], EC 2.8.3.5) is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that its catalytic mechanism involves the transient thioesterification of an active-site ... >> More

  The enzyme CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase [3-oxoacid CoA transferase], EC 2.8.3.5) is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that its catalytic mechanism involves the transient thioesterification of an active-site glutamate residue by CoA. As a means of identifying this glutamate within the sequence, we have made use of a fortuitous autolytic fragmentation that occurs at the active site when the enzyme-CoA covalent intermediate is heated. The presence of protease inhibitors has no effect on the extent of cleavage detectable by SDS-PAGE, supporting the view that this fragmentation is indeed autolytic. This fragmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the resulting C-terminal fragment is blocked at its N-terminus by a pyroglutamate moiety, it is not amenable to direct sequencing by the Edman degradation method. As an alternative, we have studied a peptide (peptide D) generated specifically by autolysis of the nicked enzyme and predicted to have an N-terminus corresponding to the site of proteolysis and a C-terminus determined by the site of autolysis. This peptide was purified by reversed-phase HPLC and subsequently characterized by electrospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conserved in all sequenced CoA transferases, is the catalytically active amino acid. This information should prove useful to future mutagenesis work aimed at better understanding the active-site structure and catalytic mechanism of CoA transferase. << Less

  Protein Sci. 3:975-981(1994) [PubMed] [EuropePMC]