Phosphorylated and dephosphorylated structures of pig heart, GTP-specific succinyl-CoA synthetase

Succinyl-CoA synthetase (SCS) catalyzes the reversible phosphorylation/dephosphorylation reaction:???rm succinyl ?hbox ?-?CoA+NDP+P_i?leftrightarrow succinate+CoA+NTP??where N denotes adenosine or guanosine. In the course of the reaction, an essential histidine residue is transiently phosphorylated. We have crystallized and solved the structure of the GTP-specific isoform of SCS from pig heart (EC 6.2.1.4) in both the dephosphorylated and phosphorylated forms. The structures were refined to 2.1 A resolution. In the dephosphorylated structure, the enzyme is stabilized via coordination of a phosphate ion by the active-site histidine residue and the two "power" helices, one contributed by each subunit of the alphabeta-dimer. Small changes in the conformations of residues at the amino terminus of the power helix contributed by the alpha-subunit allow the enzyme to accommodate either the covalently bound phosphoryl group or the free phosphate ion. Structural comparisons are made between the active sites in these two forms of the enzyme, both of which can occur along the catalytic path. Comparisons are also made with the structure of Escherichia coli SCS. The domain that has been shown to bind ADP in E. coli SCS is more open in the pig heart, GTP-specific SCS structure.     

Nucleotide recognition in the ATP-grasp protein carbamoyl phosphate synthetase

Synthesis of carbamoyl phosphate by carbamoyl phosphate synthetase (CPS) requires the coordinated utilization of two molecules of ATP per reaction cycle on duplicated nucleotide-binding sites (N and C). To clarify the contributions of sites N and C to the overall reaction, we carried out site-directed mutagenesis aimed at changing the substrate specificity of either of the two sites from ATP to GTP. Mutant design was based in part on an analysis of the nucleotide-binding sites of succinyl-CoA synthetases, which share membership in the ATP-grasp family with CPS and occur as GTP- and ATP-specific isoforms. We constructed and analyzed Escherichia coli CPS single mutations A144Q, D207A, D207N, S209A, I211S, P690Q, D753A, D753N, and F755A, as well as combinations thereof. All of the mutants retained ATP specificity, arguing for a lack of plasticity of the ATP sites of CPS with respect to nucleotide recognition. GTP-specific ATP-grasp proteins appear to accommodate this substrate by a displacement of the base relative to the ATP-bound state, an interaction that is precluded by the architecture of the potassium-binding loop in CPS. Analysis of the ATP-dependent kinetic parameters revealed that mutation of several residues conserved in ATP-grasp proteins and CPSs had surprisingly small effects, whereas constructs containing either A144Q or P690Q exerted the strongest effects on ATP utilization. We propose that these mutations affect proper movement of the lids covering the active sites of CPS, and interfere with access of substrate.     

Crystallization of succinyl-CoA synthetase from Escherichia coli

Well formed, tetragonal prisms of succinyl-CoA synthetase from Escherichia coli have been crystallized at room temperature from ammonium sulfate and mixtures of sodium and potassium phosphates. A systematic survey of the conditions for crystallization of the enzyme has been carried out. This has shown the addition of a small amount of an organic solvent (acetone, 2-methyl-2,4-pentanediol, tert-butyl alcohol, or tertamyl alcohol) to the phosphate media and of CoA to the sulfate media to be beneficial in producing large, single crystals suitable for analysis by x-ray diffraction methods. Preliminary examination of precession photographs reveals that the crystals from phosphate media have a unit cell of symmetry P4222 with dimensions a = b = 94 A and c = 248 A. Evidence suggests that there may be only half of the (alpha beta)2 tetramer/asymmetric unit in these crystals. The crystals from ammonium sulfate media have unit cell dimensions of a = b = 99 A and c = 399 A, a space group of P4122 (P4322), and one tetramer/asymmetric unit. They diffract to a resolution of 3.4 A. Both crystal types have large solvent contents of about 65% of the unit cell volumes. A parameter called "quality index" is introduced to facilitate comparison of crystals grown under a variety of conditions with respect to their quality of x-ray diffraction.     

Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase

The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Although analysis of residues inside the nucleotide-binding site suggests that Blastocystis SCS is GTP-specific, we demonstrate that it is ATP-specific. Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis SCS active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity. It has been proposed that residues in direct contact with the substrate determine nucleotide specificity in SCS. However, our results indicate that, in Blastocystis , an electrostatic gatekeeper controls which ligands can enter the binding site.     

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

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.     

Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.     

Function of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes

Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis.     

Partial purification and characterization of succinyl-CoA synthetase from Saccharomyces cerevisiae

Succinyl-CoA synthetase from Saccharomyces cerevisiae was partially purified (20-fold) with a yield of 44%. The Michaelis-Menten constants were determined: Km (succinate) = 17 mM; Km (ATP) = 0.13 mM; Km (CoA) = 0.03 mM. The succinyl-CoA synthetase has a molecular weight of about 80000 dalton (as determined by polyacrylamide gradient gel electrophoresis). The pH optimum is at 6.0. During fermentation the activity of succinyl-CoA synthetase is lower than in aerobically grown yeast cells. The presence of succinyl-CoA synthetase in fermenting yeasts may be regarded as an indication for the oxidative formation of succinate. In fermenting yeast cells succinyl-CoA synthetase is repressed by glucose if ammonium sulphate serves as nitrogen source. This catabolite repression is not observed with disaccharides or when amino acids are used as nitrogen source.     

A novel effect of vanadium ions: inhibition of succinyl-CoA synthetase

Effect of vanadate and vanadyl ions on the ATP-dependent succinyl-CoA synthetase (A-SCS) solubilized by Lubrol-PX from the rat brain mitochondria was tested. Vanadate added to the assay medium at 10(-5) mol.l-1 and 10(-4) mol.l-1 concentrations inhibited the enzyme activity by about 50% and 94%, respectively. When the enzyme was solubilized from the mitochondria preincubated with 10(-4) mol.l-1 and 10(-3) mol.l-1 vanadate, the residual inhibitions were 55% and 100% respectively. The vanadyl cation also induced inhibition of the A-SCS activity but the effect was less expressed. At 10(-4) mol.l-1 concentration only 20% inhibition was achieved. The A-SCS solubilized from the mitochondrial subfractions (perikaryal, light and heavy synaptosomal) differed neither in the activity of A-SCS nor in the susceptibility toward action of vanadium ions. A strong dependence of the vanadate inhibition on the concentration of succinate was observed. The above effect (50% inhibition) could be demonstrated only at saturating concentration of succinate (50 mmol.l-1). The mechanism of vanadium ions action as well as differences between vanadate and vanadyl ions effects are discussed.     

Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5''-p-fluorosulphonylbenzoyladenosine.

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). Optimal inactivation by 5'-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5'-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5'-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5'-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5'-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5'-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5'-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.     

Cloning and expression of the succinyl-CoA synthetase genes of Escherichia coli K12

The genes encoding both subunits of the succinyl-CoA synthetase of Escherichia coli have been identified as distal genes of the suc operon, which also encodes the dehydrogenase (Elo; sucA) and succinyltransferase (E2o; sucB) components of the 2-oxoglutarate dehydrogenase complex. The newly defined genes express polypeptides of 41 kDa (sucC) and 31 kDa (sucD), corresponding to the beta and alpha subunits of succinyl-CoA synthetase, respectively. The genes are thus located at 16.8 min in the E. coli linkage map, together with the citrate synthase (gltA) and succinate dehydrogenase (sdh) genes, in a cluster of nine citric acid cycle genes: gltA-sdhCDAB-sucABCD. Four deletion strains lacking all of these citric acid cycle enzymes were characterized. The succinyl-CoA synthetase activities of strains harbouring plasmids containing the sucC and sucD genes were amplified some fourfold. Further enzymological studies indicated that expression of succinyl-CoA synthetase is coordinately regulated with 2-oxoglutarate dehydrogenase.     

ADP-binding site of Escherichia coli succinyl-CoA synthetase revealed by x-ray crystallography

Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction via a phosphorylated histidine intermediate (His 246alpha): succinyl-CoA + P(i) + NDP <--> succinate + CoA + NTP (N denotes adenosine or guanosine). To determine the structure of the enzyme with nucleotide bound, crystals of phosphorylated Escherichia coli SCS were soaked in successive experiments adopting progressive strategies. In the first experiment, 1 mM ADP (>15 x K(d)) was added; Mg(2+) ions were omitted to preclude the formation of an insoluble precipitate with the phosphate and ammonium ions. X-ray crystallography revealed that the enzyme was dephosphorylated, but the nucleotide did not remain bound to the enzyme (R(working) = 17.2%, R(free) = 22.8% for data to 2.9 A resolution). Catalysis requires Mg(2+) ions; hence, the "true" nucleotide substrate is probably an ADP-Mg(2+) complex. In the successful experiment, the phosphate buffer was exchanged with MOPS, the concentration of sulfate ions was lowered, and the concentrations of ADP and Mg(2+) ions were increased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP-Mg(2+) complex bound in the ATP-grasp fold of the N-terminal domain of each beta-subunit (R(working) = 19.1%, R(free) = 24.7% for data to 3.3 A resolution). We describe the specific interactions of the nucleotide-Mg(2+) complex with SCS, compare these results with those for other proteins containing the ATP-grasp fold, and present a hypothetical model of the histidine-containing loop in the "down" position where it can interact with the nucleotide approximately 35 A from where His 246alpha is seen in both phosphorylated and dephosphorylated SCS.     

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.     

Inhibition by streptozotocin of the activity of succinyl-CoA synthetase in vitro and in vivo

The activity of succinyl-CoA synthetase from mouse liver and kidney was inhibited by streptozotocin in vitro. Streptozotocin behaved essentially as a non-competitive inhibitor, and the following kinetic values were obtained (in the presence of 10 nM streptozotocin): apparent Km 1.7 mM, apparent Ki 10 nM, and kcat 440 nkat X kg-1. Compared with non-diabetic control mice, the succinyl-CoA synthetase activity was significantly decreased in the islets and kidneys of mice with early (1 h) and manifest (greater than or equal to 2 days) streptozotocin diabetes, whereas the activity in the liver was not significantly altered. Inhibited succinyl-CoA synthetase activity is believed to play a prominent role in the cellular effects of streptozotocin.     

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors. The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.     

Folding and assembly of the Escherichia coli succinyl-CoA synthetase heterotetramer without participation of molecular chaperones.

Succinyl-CoA synthetase of Escherichia coli (alpha 2B2 subunit structure) has been shown to fold and assemble without participation by molecular chaperones. Renaturation experiments showed that purified bacterial chaperone GroEL has no effect on the folding and assembly of the active tetrameric enzyme. When isolated 35S-labeled alpha or beta subunits were incubated with GroEL in the absence of ATP, there was no complex formation between the subunits and GroEL. These in vitro results were confirmed by in vivo analysis of the folding and assembly of newly synthesized succinyl-CoA synthetase subunits. When expression of the subunits was induced in E. coli strains that bear GroEL or GroES temperature-sensitive mutations, the assembly of active succinyl-CoA synthetase was not affected as the temperature was raised to 43 degrees C. These and other observations are discussed that indicate that folding and assembly of succinyl-CoA synthetase may be independent of assistance by any chaperone.