Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates.

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H+). Although its acid-base properties were indistinguishable from those of epsilon-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the epsilon-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0. It is apparent that the assay of epsilon-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236--250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H⁺). Although its acid-base properties were indistinguishable from those of δ-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the δ-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0.It is apparent that the assay of δ-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236–250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

Apparent ATP-linked succinate thiokinase activity and its relation to nucleoside diphosphate kinase in mitochondrial matrix preparations from rabbit.

The relative abilities of ATP and GTP to support succinyl-CoA synthesis by mitochondrial matrix fractions prepared from rabbit heart and liver mitoplasts were investigated. The activity supported by ATP in rabbit heart preparations was less than 15% of that obtained with GTP, while no ATP-supported activity was observed in rabbit liver preparations. However, the addition of 30 micromolar GDP to matrix fractions from either heart or liver stimulated the ATP-supported activity to 40% of that observed with GTP, and the further addition of bovine liver nucleoside diphosphate kinase in the presence of 8 microM added GDP increased the activity to near that observed with GTP. The specific activity of nucleoside diphosphate kinase assayed directly in mitochondrial matrix from heart was about 15% of the specific activity of ATP-supported succinate thiokinase induced upon adding GDP. Evidence for a complex between nucleoside diphosphate kinase and succinate thiokinase in mitochondrial matrix from rabbit heart was obtained by glycerol density gradient centrifugation. It is proposed that binding of nucleoside diphosphate kinase to succinate thiokinase activates the former enzyme, accounts for the ATP-supported succinyl-CoA synthetase activity observed, and is involved in the channeling of high energy phosphate from GTP produced in the Krebs cycle to the ATP pool.     

Distinct physiological roles of animal succinate thiokinases. Association of guanine nucleotide-linked succinate thiokinase with ketone body utilization

Two distinct succinate thiokinases have recently been shown to exist in animal tissues, one specific for guanine nucleotide and the other for adenine nucleotide. Their physiological roles have here been investigated by comparing the levels of the two enzymes in liver and brain of normal and diabetic rats. A marked rise in the level of brain guanine nucleotide-linked succinate thiokinase in the diabetic condition is consistent with an enhanced utilization of ketone bodies and hence with the associated elevated demand for succinyl-CoA for the activation of acetoacetate. Taken together with the reported mitochondrial values of the ATP/ADP and GTP/GDP ratios, the results are interpreted to indicate that the adenine nucleotide-linked enzyme functions as a component of the citric acid cycle whereas the guanine nucleotide-linked enzyme functions in the opposite metabolic direction to produce succinyl-CoA from succinate.     

Enzyme and Nucleic Acid Formation During Synchronous Growth of Rhodopseudomonas spheroides

The synthesis of various cell components was examined during the anaerobic photosynthetic growth of synchronous populations of Rhodopseudomonas spheroides. Net deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein increased continuously as did the rate of incorporation of radioactive precursors into protein. The rates of incorporation of radioactive precursors into RNA and DNA were marked by abrupt discontinuities. It is not clear whether these discontinuities represent changes in rates of synthesis or fluctuations in precursor pools. Although the synthesis of bacteriochlorophyll occurred in a continuous manner, those enzymes examined which are involved in the synthesis of tetrapyrroles, i.e., succinyl CoA thiokinase, delta-aminolevulinic acid synthetase, and delta-aminolevulinic acid dehydrase, increased discontinuously. Two other enzymes not involved in tetrapyrrole biosynthesis were examined. Alkaline phosphatase increased in a stepwise manner during the division cycle, whereas the synthesis of ornithine transcarbamylase increased rapidly before leveling off for a period of time until synthesis began again. In each instance of discontinuous enzyme synthesis, increases occurred at regular and characteristic times during the division cycle. Ammonium sulfate precipitation was employed to remove low molecular weight end product inhibitors from enzyme preparations. These studies suggested that the stepwise increases in enzyme activity observed in the present investigation were not affected by periodic end product inhibition. A temporal map of enzyme synthesis during the division cycle was constructed. Both delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydrase appeared early in the division cycle, whereas alkaline phosphatase and succinyl CoA thiokinase appeared later on.     

Association between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase

The kinetic parameters of the individual reaction of pig heart alpha-ketoglutarate dehydrogenase complex, succinate thiokinase and the alpha-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The KCoAm of alpha-ketoglutarate dehydrogenase complex and the K-succinyl CoAm of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with alpha-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and alpha-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.     

The NMR study of succinate formation from exogenous precursors in anaerobic rat heart mitochondria

Succinate formation during incubation of isolated rat heart mitochondria with exogenous precursors, malate, alpha-ketoglutarate, oxaloacetate and L-glutamate was studied in the absence of aeration. The formation of succinate, the end product of the tricarboxylic acid cycle, occurs via two pathways: through reduction of oxaloacetate or malate and via oxiation of alpha-ketoglutarate. The highest rate of succinate synthesis was observed when mitochondria were incubated with a mixture of 5 mM L-glutamate and 10 mM oxaloacetate, i.e., when both routes were used simultaneously. The [U-13C]succinate/succinate and aspartate/succinate ratios were equal to 2, when mitochondria were incubated with 5 mM [U-13C]glutamate and 10 mM oxaloacetate. Therefore, the amount of succinate formed from [13C]alpha-ketoglutarate via transamination of [13C]glutamate with oxaloacetate exceeds twice succinate production from oxialoacetate. These data suggest that GTP formation in the succinic thiokinase reaction should exceed twice the ATP yield coupled with NADH-dependent reduction of fumarate.     

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse,rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic δ-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim. Biophys. Acta (1970)208, 236–250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of δ-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than δ-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F^?, malonate, and arsenite), only F^? (100 mm final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freezethawing of fresh homogenates just before assay did not increase the rate of δ-aminolevulinic acid formation.     

Two distinct succinate thiokinases in both bloodstream and procyclic forms of Trypanosoma brucei

Two succinate thiokinase activities specific for either adenine or guanine nucleotides have been found in Trypanosoma brucei. Key glycolytic and citric acid cycle enzymes were measured to show repression of glycolysis and derepression of the citric acid cycle in the procyclic form, relative to the bloodstream form. A marked rise in adenine-linked succinate thiokinase activity accompanied a rise in activity of citric acid cycle enzymes. However, guanine-linked succinate thiokinase was found to increase only slightly in activity. These results implicate the adenine-linked enzyme as an essential component of the citric acid cycle, whereas the guanine-linked enzyme appears to be under separate control. This communication also reports for the first time the occurrence of citrate synthase activity in the bloodstream (long slender) form of T. brucei.     

Inactivation of Escherichia coli succinic thiokinase by selective oxidation of thiol groups by permanganate

Succinic thiokinase from $\text{Escherichia coli}$ was rapidly inactivated by permanganate ion at 25° and 0°. On the basis of the cysteic acid content of hydrolysates of treated protein, oxidation of 3 sulfhydryl groups appeared to effect total loss of thiokinase activity. However, titration of the same protein samples revealed that 4 important sulfhydryl groups (a fraction of which was possibly in disulfide form) were more likely oxidized during the inactivation process. Significant protection of the enzyme against permanganate inactivation was obtained by the following additions: ATP-Mg²⁺ and succinate (51%); desulfo-CoA alone (53%); and ATP-Mg²⁺, succinate, and desulfo-CoA (93%). No protection was observed when either inorganic phosphate or arsenate was added.     

The activities of the citric acid-cycle enzymes in rat bone-marrow cells

The activities of the eight citric acid-cycle enzymes of rat bone-marrow cells were determined along with several other mitochondrial and non-mitochondrial enzymes. Four of the citric acid-cycle enzymes (aconitase, succinyl-CoA thiokinase, α-oxoglutarate dehydrogenase and succinate dehydrogenase) have closely similar low activities; two [isocitrate dehydrogenase (NAD) and citrate synthase] have intermediate activities; the remaining two (malate dehydrogenase and fumarase) have high activities. The other enzymes surveyed also exhibited a spread of three orders of magnitude, the mitochondrial enzymes showing no less variation than the others.     

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.     

Delta-aminolevulinic acid synthetase assay in chicken liver homogenates and particulate fractions.

Various assays for δ-aminolevulinic acid synthetase in chicken liver homogenates and particulate fractions were studied. The assay methods fall into two groups, those using exogenous succinyl-CoA generating systems and those depending on endogenous succinyl-CoA formation. In the former, the native samples showed low activity and a poor relationship between protein concentration and activity. Sonication of the samples was required to obtain higher activity and a linear relationship between protein concentration and activity. The primary factor limiting the full expression of the enzyme activity in these samples was thought to be the permeability barrier of mitochondrial membranes. In the sonicated samples the assay is limited to low protein concentrations. The addition of 100 mm sodium or potassium fluoride to the assay made possible the use of higher protein concentrations. Fluoride probably exerts its effect by preventing the rapid destruction of ATP by ATPase and providing enough ATP for the succinyl-CoA generating system. This fluoride effect was observed in the sonicated homogenates and particulate fractions of chick embryo, chick and adult chicken livers and cultured chick embryo liver cells. In those assays depending on the endogenous formation of succinyl-CoA the native homogenates and particulate fractions had relatively low δ-aminolevulinic acid synthetase activity and sonication or the addition of fluoride had no enhancing effect.     

Channeling of TCA cycle intermediates in cultured Saccharomyces cerevisiae

Oxidation of [3-13C]propionate was studied in cultured yeast cells, and the distribution of label in the 2- and 3-positions of alanine was detected by 13C NMR. [3-13C]Propionate forms [2-13C]succinyl-CoA in the mitochondria which then enters the citric acid cycle and forms malate through two symmetrical intermediates, succinate and fumarate. If these symmetrical intermediates randomly diffuse from one enzyme to the next in mitochondria as is normally assumed, then 13C labeling in malate C2 and C3 must be equal. However, any direct transfer of metabolites from site to site between succinate thiokinase, succinate dehydrogenase, and fumarase would result in an uneven distribution of 13C in malate C2 and C3 and any molecules derived from malate. Since pyruvate may be derived from malate via the malic enzyme and subsequently converted into alanine by transamination, any 13C asymmetry in alanine C2 and C3 must directly reflect the 13C distribution in the malate pool. During oxidation of [3-13C]propionate, we detect a significant quantity of labeled alanine, where 13C enrichment in C3 is significantly higher than that in C2. Inhibition of succinate dehydrogenase with malonate or creating conditions that increase the chances of a back-reaction (from malate to fumarate) result in a significant decrease in the asymmetric labeling of alanine. Ubiquinone-deficient yeast cells (having only 10% of the oxidative capacity of wild-type cells) could slowly oxidize propionate, but in this case the 13C labeling was equal in the C2 and C3 of alanine, showing that isotope randomization had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

sucAB and sucCD are mutually essential genes in Escherichia coli

sucAB and sucCD of Escherichia coli encode enzymes that generate succinyl-CoA from 2-oxoglutarate and succinate, respectively. Their mutual essentiality was studied. sucAB and sucCD could be deleted individually, but not simultaneously. The mutual essentiality of sucAB and sucCD was further confirmed by the conditional expression of sucABCD, sucAB, and sucCD under the control of a P(BAD) in E. coli MG1655, E. coli MG1655 (DeltasucCD), and E. coli MG1655 (DeltasucAB), respectively. These strains grew well in Luria-Bertani medium containing 0.1% arabinose, but not in the absence of arabinose unless the medium was supplemented with succinyl-CoA. Our results indicate that either sucAB or sucCD is enough to produce succinyl-CoA that is essential for cell viability.     

琥珀酸脱氢酶或琥珀酰辅酶A合成酶缺失促进大肠杆菌积累5-氨基乙酰丙酸

5-氨基乙酰丙酸 (ALA) 是生物体内四吡咯类化合物的合成前体,在农业及医药领域应用广泛,是极具开发价值的高附加值生物基化学品。目前利用外源C4途径的重组大肠杆菌发酵生产ALA的研究主要利用LB培养基并添加葡萄糖和琥珀酸、甘氨酸等合成前体,成本较高。琥珀酸在C4途径中以琥珀酰辅酶A的形式直接参与ALA的合成。文中在以葡萄糖为主要碳源的无机盐培养基中研究了琥珀酰辅酶A下游代谢途径琥珀酸脱氢酶编码基因sdhAB和琥珀酰辅酶A合成酶编码基因sucCD缺失对ALA积累的影响。与仅表达异源ALA合成酶的对照菌株相比,sdhAB和sucCD缺失菌株ALA的产量分别提高了25.59%和12.40%,且ALA的积累不依赖于琥珀酸的添加和LB培养基的使用,从而大幅降低了生产成本,显示出良好的工业应用前景。     

A spectrophotometric assay for deacetoxycephalosporin C synthase

A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. Km values of 0.18 mM for penicillin N and 0.16 mM for alpha-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.     

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

Tricarboxylic acid cycle enzymes following thiamine deficiency

Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the alpha-ketoglutarate dehydrogenase complex (KGDHC) (-21.5%) and PDHC (-10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase (STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter brain function in neurodegenerative disorders.     

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.