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.     

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.     

Studies of the process of renaturation and assembly of Escherichia coli succinyl-CoA synthetase from its alpha and beta subunits

Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation step of the tricarboxylic acid cycle. The enzyme, as isolated from Escherichia coli, has an alpha 2 beta 2 subunit structure. It is known that substrate-binding sites are distributed between both subunit types and that the active enzyme is the nondissociating tetramer. This paper describes a study of the process of assembly of the enzyme from its denatured constituent subunits. Starting with equimolar mixtures of the subunits that are prepared in denaturing conditions (6 M urea, 5% acetic acid), rapid renaturation to produce virtually a fully active enzyme occurs after neutralization and dilution under suitable conditions. This process occurs most efficiently in the presence of either ATP or Pi, indicating that occupation of the phosphoryl-binding site on the refolding alpha subunit facilitates productive intrasubunit interactions. We have determined conditions of protein concentration, pH, temperature, final urea concentration, and buffer compositions that optimize both the rate and extent of production of active enzyme. The final refolded product is indistinguishable from the native species with respect to its specific catalytic activity, size, and other physical properties. To probe further the mechanism and route of renaturation, we have shown that the rate of appearance of activity has first-order dependence on each of the two subunits. The step that determines the rate of assembly is thus bimolecular, such as the association of structural monomers to form a dimeric transient species. The highly specific mutual interactions between the refolding transient species of subunits must be essential for the correct assembly of this enzyme from the two gene products in vivo.     

Folding and assembly of oligomeric proteins in Escherichia coli.

High levels of expression of oligomeric proteins in heterologous systems are frequently associated with misfolding and accumulation of the polypeptides in inclusion bodies. This reflects aspects of the folding and assembly pathways of oligomeric proteins, which generally proceed from either folding intermediates or native-like metastable species that are not in their final conformation. Methods for optimizing the yield of correctly assembled oligomers are discussed.     

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.     

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.     

Affinity purification and characterization of the Escherichia coli molecular chaperones

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB). Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized beta-casein (30 mg/g beads) column. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP. The cold water and Mg-ATP eluates were analyzed by SDS-PAGE. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB. The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented.     

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.     

Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. beta-Cys325 is a nonessential active site residue

Chemical modification experiments have shown that sulfhydryl groups play an important role in the mechanism of action of Escherichia coli succinyl-CoA synthetase. One of these sulfhydryl groups has been localized in the beta-subunit of the enzyme using the coenzyme A affinity analog, CoA disulfide-S,S-dioxide (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4943). Recently, it has been shown that the reactive sulfhydryl group resides in Cys325 (Nishimura, J. S., Mitchell, T., Ybarra, J., and Matula, J. M., submitted to Eur. J. Biochem. for publication). In the present study, we have changed Cys325 to a glycine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is 83% as active as wild type enzyme. In contrast to wild type succinyl-CoA synthetase, the mutant is refractory to chemical modification by CoA disulfide-S,S-dioxide and methyl methanethiolsulfonate. It is also less reactive with N-ethylmaleimide. Thus, beta-Cys325 is a nonessential active site residue.     

Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. Histidine 142 alpha is a facilitative catalytic residue

There are 11 histidine residues in Escherichia coli succinyl-CoA synthetase. His-246 alpha is well established as the phosphorylation site of the enzyme. Replacement of this histidine by asparagine (Mann, C. J., Mitchell, T., and Nishimura, J. S. (1991) Biochemistry 30, 1497-1503) or by aspartic acid (Majumdar, R., Guest, J. R., and Bridger, W. A. (1991) Biochim. Biophys. Acta 1076, 86-90) through site-directed mutagenesis resulted in complete loss of enzyme activity. Chemical modification experiments suggested a second histidine at the active site (Collier, G. E., and Nishimura, J. S. (1979) J. Biol. Chem. 254, 10925-10930). In the present study, we have changed His-142 alpha to an asparagine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is practically devoid of enzyme activity but can be thiophosphorylated with adenosine 5'-O-(thiotriphosphate) and dethiophosphorylated with ADP at rates that are significantly faster than those with wild type enzyme. The observation that phosphorylated mutant enzyme can be dephosphorylated with succinate and with succinate plus desulfo-CoA at rates comparable with those with wild type enzyme suggests that mutant enzyme can bind succinate and CoA. Dethiophosphorylation of the enzyme in the presence of CoA plus succinate proceeds much faster with wild type than with mutant. While there was no significant change in KCoA or Ksuccinate, the turnover number for dethiophosphorylation of the mutant was 10-fold lower. These data are consistent with location of His-142 alpha at the active site and a facilitative role for this residue in catalysis.     

Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity

Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Adenosine 5'-tetraphosphate is synthesized by the histidine alpha 142----asparagine mutant of Escherichia coli succinyl-CoA synthetase.

Recently, we described the properties of a mutant (H142N) of Escherichia coli succinyl coenzyme A (CoA) synthetase in which His-142 of the alpha-subunit was changed to Asn (Luo, G.-X., and Nishimura, J.S. (1991) J. Biol. Chem. 266, 20781-20785). The mutant enzyme was practically devoid of ability to catalyze the overall reaction but was able to catalyze half-reactions at significant rates. Thus, phosphorylation by ATP and dephosphorylation by ADP of the mutant enzyme occurred at rates that were at least 10 times greater than those with wild type enzyme, and dephosphorylation by succinate plus CoA (succinyl-CoA formation) proceeded with a Vmax of 10% that of wild type, with no change in Km for succinate and very little change in Km for CoA. In the present work, it has been shown that incubation of 32P-labeled H142N with ATP caused a rapid depletion of label from the enzyme and incorporation of radioactivity into a nucleotide species that was neither ATP nor ADP. This reaction was catalyzed at comparatively negligible rates by wild type enzyme. Analysis of the labeled product by high pressure liquid chromatography and 31P NMR revealed that it was adenosine 5'-tetraphosphate (AP4). Incubation of labeled H142N with the ATP analog beta,gamma-methylene adenosine triphosphate also gave a product that appeared to be the corresponding tetraphosphate. The reaction in which AP4 was formed was greatly stimulated by the addition of phosphoenolpyruvate plus pyruvate kinase and strongly inhibited by ADP and by CoA plus succinate. The results are consistent with binding of ATP to, and reaction with, phosphorylated succinyl-CoA synthetase to form AP4. In this reaction, it was determined that the Km for ATP and the turnover number of phosphorylated enzyme were 14.5 microM and 0.024 s-1, respectively.     

Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.     

Anaerobic growth defects resulting from gene fusions affecting succinyl-CoA synthetase in Escherichia coli K12

Summary Insertion of the fusion-generating phage Mud1 (Ap, lacZ) yielded two similar isolates, DC511 and DC512, which were unable to grow aerobically on acetate or alphaketoglutarate but which could use succinate, malate, fumarate, glycerol, and various sugars. These mutants were unable to grow anaerobically on most sugars unless provided with methionine, lysine, and delta-aminolevulinic acid, all of which require succinyl-CoA for their synthesis. The insertions of both mutants mapped at 17 min, in the suc operon. Enzyme assays indicated a lack of succinyl-CoA synthetase; however, full activity of the alpha-ketoglutarate dehydrogenase was retained. Beta-galactosidase expression by strains containing these gene fusions was reduced under anaerobic conditions. In aerobically grown cultures, both fusions were induced about fivefold in the presence of acetate. This type of regulation would be expected of a Krebs cycle enzyme.