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.     

Adenosine 5'-O-(3-thio)triphosphate, a substrate and potent inhibitor of Escherichia coli succinyl-CoA synthetase. Additional evidence for a cooperative alternating-sites mechanism

Adenosine 5'-O-(3-thio)triphosphate (ATP gamma S) has been shown to be a potent inhibitor of Escherichia coli succinyl-CoA synthetase. This inhibition was competitive with respect to ATP and GTP (Ki values of 0.8 and 0.7 microM, respectively) and mixed with respect to CoA and succinate. ATP gamma S previously had been shown to be a weak substrate of the enzyme, probably because of the relatively sluggish reactivity of the thiophosphoryl enzyme intermediate (Wolodko, W. T., Brownie, E. R., O'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119). In our work, reaction of thiophosphoryl enzyme with ADP was greatly stimulated by succinyl-CoA, an observation that is consistent with the concept of alternating-sites cooperativity. Thiophosphoryl group release did not appear to be accompanied by "other-site" phosphorylation, in contrast to ATP stimulation of thiophosphoryl group release in the presence of succinate and CoA (Wolodko et al., see above). In addition, ADP did not appear to be required in the latter reaction.     

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.     

Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia coli succinyl-CoA synthetase

Succinyl-CoA synthetase (EC 6.2.1.5, succinate: CoA ligase (ADP-forming)) of Escherichia coli is an α₂β₂ tetramer, with the active site believed to be located at the point of contact between the two subunit types. It has been previously established that the reaction involves the intermediate participation of a phosphorylated enzyme form in the process of catalysis. The site of phosphorylation (His-246) and the binding sites for the substrates ADP and ATP are located in the α subunit, and the succinate and CoA binding sites are in β. A mutant form of this enzyme, with the active site histidine residue replaced by aspartate, has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subunit assembly, but has no ability to catalyze the overall reaction. As expected, the His-246 α →Asp mutant is incapable of undergoing phosphorylation. We have developed an assay based upon the arsenolysis of succinyl-CoA that effectively isolates the partial reaction that occurs in the portion of the active site contributed by the β subunit; this reaction does not involve covalent participation of His-246 α. We have found that the His-246 α →Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His-246 α is required for the establishment of the microenvironment in this portion of the active site that is required for the corresponding step of the overall reaction.     

Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation.

Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E. coli succinyl-coenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme. This is true when either ATP or succinyl-CoA and Pi are used to phosphorylate the enzyme. The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site. GDP also alters the phosphorylation of the E. coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor. GDP appears to function by neutralizing the action of a specific inhibitory protein. This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism. These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands.     

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.     

Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei

Oxidative phosphorylation and substrate level phosphorylation catalyzed by succinyl-CoA synthetase found in the citric acid and the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle contribute to mitochondrial ATP synthesis in procyclic Trypanosoma brucei. The latter pathway is specific for trypanosome but also found in hydrogenosomes. In organello ATP production was studied in wild-type and in RNA interference cell lines ablated for key enzymes of each of the three pathways. The following results were obtained: 1) ATP production in the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle was directly demonstrated. 2) Succinate dehydrogenase appears to be the only entry point for electrons of mitochondrial substrates into the respiratory chain; however, its activity could be ablated without causing a growth phenotype. 3) Growth of procyclic T. brucei was not affected by the absence of either a functional citric acid or the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle. However, interruption of both pathways in the same cell line resulted in a growth arrest. In summary, these results show that oxygen-independent substrate level phosphorylation either linked to the citric acid cycle or tied into acetate production is essential for growth of procyclic T. brucei, a situation that may reflect an adaptation to the partially hypoxic conditions in the insect host.     

Two glutamate residues, Glu 208 alpha and Glu 197 beta, are crucial for phosphorylation and dephosphorylation of the active-site histidine residue in succinyl-CoA synthetase.

Succinyl-CoA synthetase catalyzes the reversible reaction succinyl-CoA + NDP + P(i) <--> succinate + CoA + NTP (N denoting adenosine or guanosine). The enzyme consists of two different subunits, designated alpha and beta. During the reaction, a histidine residue of the alpha-subunit is transiently phosphorylated. This histidine residue interacts with Glu 208 alpha at site I in the structures of phosphorylated and dephosphorylated Escherichia coli SCS. We postulated that Glu 197 beta, a residue in the nucleotide-binding domain, would provide similar stabilization of the histidine residue during the actual phosphorylation/dephosphorylation by nucleotide at site II. In this work, these two glutamate residues have been mutated individually to aspartate or glutamine. Glu 197 beta has been additionally mutated to alanine. The mutant proteins were tested for their ability to be phosphorylated in the forward or reverse direction. The aspartate mutant proteins can be phosphorylated in either direction, while the E208 alpha Q mutant protein can only be phosphorylated by NTP, and the E197 beta Q mutant protein can only be phosphorylated by succinyl-CoA and P(i). These results demonstrate that the length of the side chain at these positions is not critical, but that the charge is. Most significantly, the E197 beta A mutant protein could not be phosphorylated in either direction. Its crystal structure shows large differences from the wild-type enzyme in the conformation of two residues of the alpha-subunit, Cys 123 alpha-Pro 124 alpha. We postulate that in this conformation, the protein cannot productively bind succinyl-CoA for phosphorylation via succinyl-CoA and P(i).     

Subunit interaction during catalysis. ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction

A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase. This is based on the extent of oxygen exchange between medium [18O]Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis. Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium. With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered. In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant. Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed. These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange. Measurement of the distribution of [18O]Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations. These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site.     

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.     

Thiophosphorylation as a probe for subunit interactions in Escherichia coli succinyl coenzyme A synthetase. Further evidence for catalytic cooperativity and substrate synergism

Succinyl-CoA synthetase has an (alpha beta)2 subunit structure and shows half-of-the-sites reactivity with respect to the formation of the phosphohistidyl residues that acts as a catalytic intermediate. Adenosine 5'-O-(3-thio)triphosphate has been found to be a substrate, but the overall maximum velocity is 3 orders of magnitude lower than that seen with ATP. Moreover, steps of the reaction involving thiophosphoryl transfer are much slower than the corresponding phosphoryl transfers. These properties of adenosine 5'-O-(3-thio)triphosphate as a substrate have been exploited to test the concept of alternating sites catalytic cooperativity proposed earlier as a rationale for the subunit structure of succinyl-CoA synthetase. As predicted by this model for catalysis, the rate of discharge of thiophosphate from the enzyme in the presence of succinate and CoA is stimulated by ATP. Neither of two nonhydrolyzable analogs of ATP has an equivalent effect. The results indicate that the transfer of the thiophosphoryl group from the enzyme to succinate at one active site is not favored until the neighboring active site is phosphorylated by ATP, with accompanying reciprocal changes in the conformations of the two halves of the enzyme molecule.     

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.     

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.     

Reaction of substrates with 35S-thiophosphorylated succinyl-CoA synthetase of pig heart. Similarities to the case of the Escherichia coli enzyme

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) was found to be a substrate of pig heart succinyl-CoA synthetase with Km and kcat values of 3 microM and 0.23 s-1, respectively. The corresponding values with GTP as substrate were 48 microM and 65 s-1. 35S-thiophosphorylated enzyme was prepared by incubation of pig heart succinyl-CoA synthetase with [35S]GTP gamma S. A comparison was made of thiophosphoryl group release by substrates from this alpha beta (one active site) enzyme with that of the alpha 2 beta 2 (two active sites) Escherichia coli enzyme (Wolodko, W. T., Brownie, E. R., O'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119; Nishimura, J. S., and Mitchell, T. (1984) J. Biol. Chem. 259, 9642-9645). It was found, as in the case of the E. coli enzyme, that thiophosphoryl group release by GDP and by succinate plus CoA was stimulated by succinyl-CoA and GTP, respectively. The same result was observed at 1, 0.1, and 0.01 mg/ml, lending assurance that these phenomena were not exhibited by an aggregated form of the pig heart enzyme. While an alternating-sites catalytic cooperativity model is not ruled out for the E. coli enzyme, it is proposed that the NTP- and succinyl-CoA-stimulated release of thiophosphoryl groups from either enzyme involves a "same-site" mechanism, to be distinguished from an "other-site" mechanism.     

Reaction mechanism and structural model of ADP-forming Acetyl-CoA synthetase from the hyperthermophilic archaeon Pyrococcus furiosus: evidence for a second active site histidine residue

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P(i) acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the beta-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (alpha(2)beta(2)) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His-71beta. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:     

Fluorometric assays for succinyl-CoA and propionyl-CoA in mitochondrial extracts

Fluorometric assay procedures are described for the quantitative measurements of succinyl-CoA and propionyl-CoA down to concentrations of 0.1 μm in the reaction mixture. The enzymatic assay for succinyl-CoA couples the reaction of 3-ketoacid CoA transferase (succinyl-CoA transferase) to β-OH butyryl-CoA dehydrogenase. A simple purification procedure is described for the isolation of succinyl-CoA transferase from beef heart. Two enzyme assays for propionyl-CoA are described. In the first, CoA, acetyl-CoA and propionyl-CoA are assayed by sequential addition of α-ketoglutarate dehydrogenase, citrate synthase and phosphotransacetylase. The second assay for propionyl-CoA utilized propionyl-CoA carboxylase to convert propionyl-CoA to methylmalonyl-CoA in the presence of ATP and bicarbonate, and the ADP formed was assayed by coupling pyruvate kinase with lactate dehydrogenase. Illustrations are given for the application of these assay procedures to measurements of succinyl-CoA and propionyl-CoA in neutralized perchloric acid extracts prepared from rat heart and liver mitochondria incubated under a variety of conditions.     

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.     

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.     

The phosphorylation of intramitochondrial AMP: A suggestion for the compartmentation of endogenous Pi pool

1. The mitochondrial level of AMP gradually diminishes during incubation of mitochondria with glutamate but does not with succinate. This decline of AMP, associated with stoichiometric increase in ADP and/or ATP, is accelerated by the addition of electron acceptors or 2,4-dinitrophenol, while arsenite, arsenate and rotenone are inhibitory. These results are in agreement with the view that AMP is phosphorylated to ADP in the inner space of rat liver mitochondria via succinyl-CoA synthetase (succinate: CoA ligase (GDP), EC 6.2.1.4) and GTP:AMP phosphotransferase dependent on the oxidation of 2-oxoglutarate, which is promoted by the transfer of electron from NADH to the respiratory chain.2. Studies of the periodical changes of chemical quantities of adenine nucleotides as well as of their labelling with ³²P_i reveals the following characteristics concerning mitochondrial phosphorylation. (i) In contrast to the mass action ratio of ATP to ADP, the ratio of ADP to AMP is not affected by the intramitochondrial concentration of P_i. (ii) ³²P_i, externally added, is incorporated into ADP much more slowly than into γ-phosphate of ATP. (iii) Conversely, ATP loses its radioactivity from γ-phosphate position more rapidly than [³²P]ADP when ³²P-labelled mitochondria are incubated with non-radioactive P_i.3. In order to elucidate the above characteristic properties of phosphorylation, a hypothetical scheme is proposed which postulates the two separate compartments in the intramitochondrial pool of P_i; one readily communicates with external P_i and is utilized for the phosphorylation of ADP in oxidative phosphorylation, while the other less readily communicates with external P_i and serves as the precursor of ADP via succinyl-CoA synthetase and GTP:AMP phosphotransferase.     

The steady state concentrations of coenzyme A-SH and coenzyme A thioester, citrate, and isocitrate during tricarboxylate cycle oxidations in rabbit heart mitochondria.

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of O2 uptake, and permits the conclusion that the Km of beta oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus L-malate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 leads to 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 leads to 3 leads to 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3 1/2) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by O2 uptake.