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PubMed1994

Kinetics of the quaternary structure change of aspartate transcarbamylase triggered by succinate, a competitive inhibitor.

The quaternary structural change of Escherichia coli aspartate transcarbamylase (ATCase) was studied by time-resolved X-ray solution scattering following the binding of carbamoyl phosphate and of succinate, a competitive inhibitor of the natural substrate L-aspartate. Stopped-flow experiments at sub-zero temperatures in the presence of 30% ethylene glycol allowed us to monitor the evolution of the scattering pattern, including the characteristic scattering peak in an s (=2 sin theta/lambda) range of 0.01-0.06 A-1. The inhibitor binding promotes a quaternary structure change from the T state toward the R state, and as expected for a simple ligand binding process, ATCase remains in the R state, unlike the physiological enzyme reaction [Tsuruta, H., et al. (1990) FEBS Lett. 263, 66-68]. After equilibrium had been established, the final scattering pattern was recorded. When the succinate concentration was sufficiently high, this pattern was the same as that given by ATCase saturated with the bisubstrate analogue N-phosphonoacetyl-L-aspartate (PALA). This implies that, under cryogenic conditions, succinate and carbamoyl phosphate promote the same quaternary structure change as PALA, which is in good agreement with the crystallographic studies of Gouaux and Lipscomb [Gouaux, J.E., & Lipscomb, W.N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4205-4208]. Scattering patterns recorded during the course of the structural transition were satisfactorily reproduced by a linear combination of the initial and final patterns, suggesting that there is no significant concentration of quaternary structure intermediates between the T and R states. This is consistent with a concerted structural transition of ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Carbamoyltransferase↗
PubMed1998

Metabolism of the dimethyl ester of [2,3-(13)C]succinic acid in rat hepatocytes.

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

Alanine↗
PubMed1997

Derepressed utilization of L-malic acid and succinic acid by mutants of Pachysolen tannophilus.

Utilization of the tricarboxylic acid (TCA) cycle intermediates, L-malic acid and succinic acid, by the yeast Pachysolen tannophilus is repressed in the presence of glucose. Strains of P. tannophilus containing mutations in two hexokinases and a glucokinase were characterized for growth on glucose plus L-malic acid or succinic acid. Increased specific utilization rates of malic acid and succinic acid in the presence of glucose were observed in mutants containing a lesion in hexokinase A, an enzyme associated with catabolite repression. Such derepressed mutants may have application in winemaking in which utilization of a major grape acid, L-malic acid, is often desirable for acidity reduction.

Glucose↗
PubMed1988

Three-dimensional structure of carbamoyl phosphate and succinate bound to aspartate carbamoyltransferase.

The three-dimensional structure of the ternary complex of carbamoyl phosphate, succinate, and aspartate carbamoyltransferase (EC 2.1.3.2) has been determined to 2.6-A resolution. The binding of the phosphate of carbamoyl phosphate is similar to the binding of the phosphonate of N-(phosphonoacetyl)-L-aspartate (PALA); interacting with the carboxylates of succinate are some of the same residues that interact with the carboxylates of PALA. The amino group of carbamoyl phosphate donates hydrogen bonds to the main-chain carbonyls of residues Pro-266 and Leu-267 and the side-chain carbonyl of Gln-137. In comparing the structure of the active sites in the PALA-enzyme complex to the active sites in the carbamoyl phosphate-succinate-enzyme complex, we find that they are similar.

Aspartate Carbamoyltransferase↗
PubMed1990

Vitamin B12S-promoted model rearrangement of methylmalonate to succinate is not a free radical reaction.

To probe for free radical intermediates in the model methylmalonate to succinate rearrangements promoted by vitamin B12s, a model series with a pentenyl side chain radical trap has been devised. The control free radical, generated by tri-n-butyltin hydride treatment of bromomethyl-pentenylmalonate thioester, undergoes rapid cyclization to the six-membered ring, and, as anticipated, no succinate rearrangement product is detected. By contrast when the bromide is treated with vitamin B12s, little cyclized product is observed; the major product is the pentenyl succinate. This result demonstrates that the latter rearrangement does not follow a free radical pathway.

Chemical Phenomena↗
PubMed1957

Intracellular localization of enzymes in spleen. I. Reduced diphosphopyridine nucleotide cytochrome c reductase, cytochrome c oxidase, and succinic dehydrogenase in the rat and guinea pig.

1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl(2) layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl(2) layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals.

Animals↗
PubMed1961

Formalin fixation in the cytochemical demonstration of succinic dehydrogenase of mitochondria.

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q(10), and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5 degrees to 10 degrees C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro-BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.

Animals↗
PubMed1976

Effect of methylprednisolone sodium succinate on hypoxic heart muscle.

The ability of 30 mg/litre methylprednisolone sodium succinate (MPSS) to modify the effects of hypoxia on isolated Langendorff-perfused rat hearts was investigated. When perfused under hypoxic conditions (pO2 less than 0.8 kPa[6 mmHg]) these hearts lose intracellular enzymes, including creatine phosphokinase (CPK) and succinic dehydrogenase (SDH). The size of the extracellular space is enhanced, the cells gain Na+ and Ca2+ and lose K+, and the endogenous stores of ATP and CP are depleted. Initially the resistance to flow in the coronary circulation falls but after 75 min of hypoxic perfusion it increases so that coronary flow is reduced. MPSS failed to prevent hypoxic muscle from either gaining Na+ and Ca2+ or losing K+. It did, however, delay the release of CPK and SDH from the hypoxic muscle, prolong the phase of increased coronary flow, and decrease the rate of depletion of the energy-rich phosphate stores. MPSS potentiated the hypoxic-induced gain in Ca2+. Whilst the effects of MPSS on coronary flow and tissue Ca2+ were probably due to the steroid part of the complex, the other changes, including the protection of the ATP and CP stores and the delayed enzyme release, were probably due to the presence of the sodium succinate.

Adenosine Triphosphate↗
PubMed1996

P/O ratios reassessed: mitochondrial P/O ratios consistently exceed 1.5 with succinate and 2.5 with NAD-linked substrates.

The efficiency of ATP synthesis coupled to cell respiration, commonly referred to as the P/O ratio, has been the subject of extensive studies for more that 50 years. The general conclusion from these studies is that respiring mitochondria can convert external ADP to ATP at a maximal P/O ratio of 3 for NAD-linked substrates and 2 for succinate. However, in recent years the validity of these "integral" values has been questioned on both mechanistic and thermodynamic grounds, and a mechanistic P/O ratio of 2.5 for NAD-linked substrates and 1.5 for succinate have been concluded on the basis of experiments with isolated mitochondria. These values have been widely adopted in the scientific literature, including several recent textbooks. In this paper we report that under optimal conditions with respect to preparation and assay procedures, the P/O ratios obtained with isolated rat liver mitochondria consistently exceed 2.5 with NAD-linked substrates and 1.5 with succinate. These results, although not excluding "nonintegral" P/O ratios due to various energy-dissipating side reactions, warrant caution in accepting the reported lower values and, in general, in referring to mechanistic considerations unless the underlying molecular mechanisms are understood.

Adenosine Diphosphate↗
PubMed1980

The binding of NADH to succinic semialdehyde dehydrogenase.

The enzyme succinic semialdehyde dehydrogenase from pig brain is a tetramer composed of identical subunits of molecular weight 41000. Fluorometric titrations conducted on samples of enzyme in pyrophosphate buffer (pH 8.4) reveal the presence of two NADH binding sites characterized by a dissociation constant of 4 microM. The unusual stoichiometry of binding of NADH, i.e. two moles NADH/enzyme tetramer, as demonstrated by emission anisotropy measurements is not due to reversible association-dissociation of the oligomeric structure at pH 8.4. The results of the fluorometric titrations are consistent with a model of extreme negative cooperativity, i.e. the affinity of NADH for the enzyme is influenced by interactions between the protomers. The reaction catalyzed by succinic semialdehyde dehydrogenase is inhibited by the substrate succinic semialdehyde with a Ki of 0.09 microM which is fivefold greater than the Km value. The binding of excess substrate to the catalytic site provides a direct and simple mechanism for regulation of the rate of product formation.

Aldehyde Oxidoreductases↗
PubMed1981

Kinetic studies with rat-brain succinic-semialdehyde dehydrogenase.

A simple procedure is described that gives an approximately 100-fold purification of rat brain succinic-semialdehyde dehydrogenase with a high yield. The enzyme exhibits a relatively low Km value for succinic semialdehyde (2.5 microM) and is inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD+ (Ki = 150 microM). p-Hydroxybenzaldehyde was shown to give competitive inhibition with respect to succinic semialdehyde and uncompetitive inhibition with respect to NAD+. Initial rate studies in the presence of a fixed concentration of this inhibitor allowed a more accurate estimation of the kinetic parameters for the uninhibited reaction. The results of these studies, together with analysis of the dead-end inhibition by AMP and the effects of NAD+ and 3-acetylpyridine--adenine dinucleotide as alternative acceptors in the reaction, were consistent with the enzyme-catalysed reaction obeying a compulsory-order mechanism in which NAD+ was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it.

Aldehyde Oxidoreductases↗
PubMed1996

Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli.

Fermentative production of succinic acid from glucose by Escherichia coli was significantly increased by overexpression of phosphoenolpyruvate carboxylase. In contrast, overexpression of phosphoenolpyruvate carboxykinase had no effect. Under optimized conditions, induction of the carboxylase resulted in a 3.5-fold increase in the concentration of succinic acid, making succinic acid the major fermentation product by weight.

Escherichia coli↗
PubMed1985

Succinic acid, a metabolic by-product of Bacteroides species, inhibits polymorphonuclear leukocyte function.

Anaerobes, in particular Bacteroides spp., are the predominant bacteria present in mixed intra-abdominal infections, yet their critical importance in the pathogenicity of these infections is not clearly defined. Succinic acid, a major fatty acid by-product of Bacteroides metabolism, was tested for its effect on neutrophil function to determine whether it might play a role in enhancing the virulence of Bacteroides-containing infections. At pH 5.5 but not pH 7.0, succinic acid at concentrations commonly found in clinical abscesses profoundly inhibits in vitro neutrophil function. It virtually obliterates phagocytic killing of Escherichia coli and reduces neutrophil random migration and chemotactic response to formyl-methionyl-leucyl-phenylalanine and C5a. These effects occur in conjunction with a reduced chemiluminescent peak and delayed time to the peak. The effect on neutrophils is only partially reversible by multiple washings. These findings suggest that succinic acid may be an important Bacteroides virulence factor when present in the microenvironment of a mixed intra-abdominal infection in which concentrations are high and the pH of the medium is reduced.

Bacteroides↗
PubMed1974

Proteins of the inner membrane of Escherichia coli: identification of succinate dehydrogenase by polyacrylamide gel electrophoresis with sdh amber mutants.

The inner or cytoplasmic membrane fraction of the cell envelope of Escherichia coli was isolated by isopycnic centrifugation on sucrose gradients. The membrane proteins were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (8.5%), and up to 56 bands were resolved. Different preparations gave very similar patterns of proteins. Succinate dehydrogenase mutants (sdh) were isolated which could not grow on succinate minimal medium, although growth on fumarate was unimpaired. The protein patterns of inner membrane preparations from sdh amber mutants were compared with the wild type, and one major band was greatly reduced in the mutants. This component, which represented approximately 5% of the inner membrane protein, was restored by introducing an amber suppressor gene (supU), which also restored the Sdh(+) phenotype. The band corresponded to a protein with a molecular weight of 67,000 daltons, which is close to that for the large subunits of the succinate dehydrogenases of Rhodospirillum rubrum and beef heart mitochondria.

Bacterial Proteins↗
PubMed1997

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.

Amino Acid Sequence↗
PubMed1992

A succinate transport mutant of Bradyrhizobium japonicum forms ineffective nodules on soybeans.

Biochemical evidence has shown that dicarboxylic acids actively support symbiotic nitrogen fixation by both fast- and slow-growing Rhizobium. Mutants defective in the active uptake of succinate have been previously described only in species of the fast-growing rhizobium. This article is a report on the isolation of mutants defective in dicarboxylate transport in a slow-growing species of rhizobium, Bradyrhizobium japonicum. One of these presumptive dicarboxylate transport mutants, GTS, was characterized further. Cultured GTS was unable to accumulate [14C]succinate above background levels but possessed normal rates of malate dehydrogenase, fumarase, and hydroxybutyrate dehydrogenase activities. When inoculated onto soybeans, GTS produced a Nod+, Fix- phenotype. The bacteroids isolated from these nodules failed to accumulate labelled succinate. Electron micrographs of nodules formed by inoculation with GTS appeared normal with the exceptions of more prominent peribacteroid spaces in the infected cells and the appearance of starch granules in the noninfected cells. The phenotypical and morphological changes observed for B. japonicum are similar to those previously reported for the fast-growing species.

Biological Transport, Active↗
PubMed1996

Nutritional efficiency of succinic acid and glutamic acid dimethyl esters in colon carcinoma cells.

The dimethyl esters of succinic acid (SAD) and glutamic acid (GME) were found to be efficiently metabolized in colon carcinoma cells of the Caco-2 line. The rate of [1,4-14C]SAD and [2,3-14C]SAD conversion to radioactive acidic metabolites, CO2, amino acids, pyruvic acid, and lactic acid suggested that the catabolism of the ester-derived succinic acid occurred mainly through the sequence of reactions catalyzed by succinate dehydrogenase, fumarase, and the malic enzyme. This coincided with a marked sparing action of SAD on the utilization of D-[2-(3)H]glucose and D-[5-(3)H]glucose and generation of 14C-labeled acid metabolites, CO2, and lactic acid from D-[U-14C]glucose by the enterocytes. Likewise, the conversion of [U-14C]GME to 14C-labeled amino acids, its oxidation compared with that of [1-(14)C]GME, and the production of NH4+ in the absence or presence of GME indicated efficient catabolism of the latter ester. Like SAD, GME decreased the utilization of D-[5-(3)H]glucose and generation of 14C-labeled acidic metabolites, pyruvate, and CO2 from D-[6-(14)C]glucose, while increasing the generation of 14C-labeled amino acids from the labeled hexose. The oxidation of D-[6-(14)C]glucose was even more severely inhibited by GME. In normal rat intestinal cells, SAM, SAD, and GME also exerted a marked sparing action on D-[U-14C]glucose oxidation. The present findings suggest, therefore, that these esters could possibly be used to sustain ATP generation in intestinal cells.

Adenosine Triphosphate↗
PubMed1984

Effects of pH, calcium, and succinate on sodium citrate cotransport in renal microvilli.

Sodium-dependent citrate uptake (v) by rabbit renal brush border vesicles was studied as a function of pH and Ca2+, citrate, and succinate concentrations to evaluate the hypothesis that factors which alter renal citrate reabsorption also alter v. At pH 8 a 10-fold increment in citrate concentration (up to 3.3 mM) was required to achieve the same v observed at pH 7. This provides proof that the protonated citrates are the effective substrates for v. At higher citrate concentrations (5-10 mM) and pH 8, substrate inhibition of v occurred. Studies of the substrate concentration dependency of v at various pH levels and calcium concentrations indicated that v was competitively inhibited by citrate3-. Changes in calcium concentrations altered v by altering the protonated citrate and citrate3- concentrations; v showed countertransport with succinate. Sodium-dependent succinate uptake (vs) was not pH dependent but was more inhibited by 1 mM citrate at pH 7 than at pH 8, indicating that the protonated citrates are inhibitors of vs. However, citrate3- was also found to inhibit vs. Thus, changes in v at the brush border membrane due to the effects of pH or calcium concentration on the protonated citrate concentration and to competition between polycarboxylates may be relevant to changes in renal citrate reabsorption in vivo.

Animals↗