Activities of kreb's cycle enzymes in the flight muscles of the tsetse fly (Glossina) and the fleshfly (Sarcophaga)

The specific activities of enzymes of the tricarboxylic acid cycle have been measured in flight muscle mitochondria of tsetse flies and fleshflies. Similar levels of those enzymes which are involved in the metabolism of both carbodydrate and proline (2-oxoglutarate dehydrogenase, succinate dehydrogenase and fumarase) were found in the mitochondria of the two insects. Enzymes of the cycle not involved in the oxidation of proline (citrata synthase, aconitase, isocitrate dehydrogenase and malate dehydrogenase) had considerably lower specific activities in the tsetse fly.     

Tricarboxylic acid cycle enzymes of a pseudophyllid cestode Penetrocephalus ganapatii

Studies on the tricarboxylic acid cycle (TCA cycle) enzymes of Penetrocephalus ganapatii reveal that the TCA cycle is only partially operative, as some of the enzymes at the start of the cycle viz. citrate synthase, aconitase and isocitrate dehydrogenase are found to be low in their activities. The high activities of malate dehydrogenase and fumarase, showing affinity towards a reverse direction, indicate that the TCA cycle operates in the reverse direction resulting in the formation of fumarate. The low succinate dehydrogenase/fumarate reductase ratio suggests that ATP generation may occur at site I of the respiratory chain during the reduction of fumarate into succinate.     

Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes

To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in alpha-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth-enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in alpha-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.     

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.     

Intracellular distribution of tricarboxylic acid cycle enzymes in liver of rainbow trout Salmo gairdneri

The intracellular distribution of enzymes of the TCA cycle was investigated in liver of rainbow trout. All enzymes of the cycle apart from succinyl thiokinase were detected. Citrate synthase, alpha-ketoglutarate dehydrogenase and succinate dehydrogenase were wholly mitochondrial. Fumarase, malate dehydrogenase, aconitase and NADP-isocitrate dehydrogenase were detected in both cytosol and mitochondria.     

Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon

The majority of all proteins of a living cell is active in complexes rather than in an isolated way. These protein-protein interactions are of high relevance for many biological functions. In addition to many well established protein complexes an increasing number of protein-protein interactions, which form rather transient complexes has recently been discovered. The formation of such complexes seems to be a common feature especially for metabolic pathways. In the Gram-positive model organism Bacillus subtilis, we identified a protein complex of three citric acid cycle enzymes. This complex consists of the citrate synthase, the isocitrate dehydrogenase, and the malate dehydrogenase. Moreover, fumarase and aconitase interact with malate dehydrogenase and with each other. These five enzymes catalyze sequential reaction of the TCA cycle. Thus, this interaction might be important for a direct transfer of intermediates of the TCA cycle and thus for elevated metabolic fluxes via substrate channeling. In addition, we discovered a link between the TCA cycle and gluconeogenesis through a flexible interaction of two proteins: the association between the malate dehydrogenase and phosphoenolpyruvate carboxykinase is directly controlled by the metabolic flux. The phosphoenolpyruvate carboxykinase links the TCA cycle with gluconeogenesis and is essential for B. subtilis growing on gluconeogenic carbon sources. Only under gluconeogenic growth conditions an interaction of these two proteins is detectable and disappears under glycolytic growth conditions.     

Enzymes of the tricarboxylic acid cycle in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate pathway were investigated in adults and infective larvae of Ancylostoma ceylanicum and Nippostrongylus brasiliensis, and their activities were compared with those obtained in rat liver. A complete sequence of enzymes of the TCA cycle, with most of them showing activities quite similar to those in the rat liver homogenate, was detected in adults of both species. All the enzymes except fumarase and malate dehydrogenase were located predominantly in mitochondria where they showed a variable distribution of activities between the soluble and the membranes fractions. Malate dehydrogenase and fumarase were found in both the mitochondria and the 9,000-g supernatant fraction. Succinyl CoA synthetase, which was present in minimum activity, appeared rate limiting. Enzymes of the glyoxylate pathway, particularly isocitrate lyase, seemed to aid the functioning of the Krebs cycle by allowing the formation of succinate from isocitrate. The infective larvae of both species also were found equipped with all the enzymes of the Krebs cycle. Nonetheless, only isocitrate lyase of the glyoxylate pathway could be detected in these parasites.     

Inhibition of aconitase and fumarase by nitrogen compounds in Rhodobacter capsulatus

High levels of aconitase and fumarase activities were found in Rhodobacter capsulatus E1F1 cells cultured with nitrate as the sole nitrogen source either under light-anaerobic or dark-aerobic conditions. Both activities were strongly and reversibly inhibited in vitro by nitrite or nitric oxide, whereas nitrate or hydroxylamine showed a lower effect. Other enzymes of the tricarboxylic acids cycle such as malate dehydrogenase or isocitrate dehydrogenase were not affected by these nitrogen compounds. When growing on nitrate in the dark R. capsulatus E1F1 cells accumulated nitrite intracellularly, so that an in vivo inhibition of aconitase and fumarase could account for the strong inhibition of growth observed in the presence of nitrite under dark-aerobic conditions.Abbreviations ACO aconitase - FUM fumarase - MDH malate dehydrogenase - ICDH isocitrate dehydrogenase - TCA tricarboxylic acid     

ENZYMES OF THE TRICARBOXYLIC ACID CYCLE IN SEA URCHIN EGGS AND EMBRYOS

Changes in the activity of some enzymes of the tricarboxylic acid cycle during development of sea urchins were investigated. Unfertilized eggs showed substantial activity of citrate synthase, aconitase, NAD- and NADP-specific isocitrate dehydrogenases, fumarase and malate dehydrogenase. During development, the activity of citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase and malate dehydrogenase increases gradually, whereas the activity of fumarase remains rather constant. There is no close correlation between changes in the enzyme activity and the increase in oxygen consumption during development. Citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase are mainly localized in the mitochondrial fraction, whereas fumarase and malate dehydrogenase are present in both mitochondrial and cytosol fractions. The intracellular localization of these enzymes does not change during development. A possible mechanism for the regulation of some enzymes of the tricarboxylic acid cycle in sea urchin eggs is discussed.     

Metabolism of Yarrowia lipolytica grown on ethanol under conditions promoting the production of alpha-ketoglutaric and citric acids: a comparative study of the central metabolism enzymes

A comparative study of the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing alpha-ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.     

Acetate catabolism in the dissimilatory iron-reducing isolate GS-15.

Acetate-grown GS-15 whole-cell suspensions were disrupted with detergent and assayed for enzymes associated with acetate catabolism. Carbon monoxide dehydrogenase and formate dehydrogenase were not observed in GS-15. Catabolic levels of acetokinase and phosphotransacetylase were observed. Enzyme activities of the citric acid cycle, i.e., isocitrate dehydrogenase, 2-oxoglutarate sythase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were observed.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Enzymes of the tricarboxylic acid cycle in Obeliscoides cuniculi (Nematoda; Trichostrongylidae) during parasitic development

The specific activities of the enzymes of the tricarboxylic acid cycle; citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were determined in early fifth-stage, young and mature adult Obeliscoides cuniculi, the rabbit stomach worm. ∝-Ketoglutarate dehydrogenase activity could not be determined in any fraction. Fumarate reductase activity was found only in the mitochondrial fraction while all other enzymes, including an NADP-dependent malic enzyme were localized in the cytoplasm. Glutamate dehydrogenase, acid and alkaline phosphatase activities were also recorded. High levels of those enzymes acting in the “reversed” direction, i.e. MDH and fumarase relative to the enzymes of the “forward” direction, i.e. citrate synthase, aconitase and isocitrate dehydrogenase suggests that under anaerobic conditions a modified tricarboxylic acid cycle can operate. Some variations in specific activities were apparent as the worms matured but no qualitative differences were observed.     

Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.

The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.     

Metabolism of Yarrowia lipolytica Grown on Ethanol under Conditions Promoting the Production of α-Ketoglutaric and Citric Acids: A Comparative Study of the Central Metabolism Enzymes

A comparative study of the enzymes of tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing -ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.     

Adaptation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> to Al-Citrate: Involvement of Tricarboxylic Acid and Glyoxylate Cycle Enzymes and the Influence of Phosphate

The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles. While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase. On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity. This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles. These observations were further confirmed by ¹H NMR and ¹³C NMR spectroscopy. No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1). This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.     

Thermostability of enzymes of the tricarboxylic acid cycle ofBacillus coagulans

The thermostability of four enzymes of the tricarboxylic acid cycle has been studied in the facultative thermophile,Bacillus coagulans. Although isocitrate dehydrogenase appeared to be more temperature-sensitive in whole-cell extracts of cultures grown at 30°C compared with that in cultures grown at 55°C, this difference could be largely eliminated by the removal of cell-wall material. The specific activity of each of the enzymes examined was approximately threefold higher in cultures grown at 55°C than in those grown at 30°C. The maximum temperature, Arrhenius plot and effect of stabilizing agents for each enzyme were examined and found to be independent of growth temperature. Sodium chloride (10% w/v) was an effective protective agent for fumarase, aconitase and malate dehydrogenase. Protection from thermal denaturation of isocitrate dehydrogenase, aconitase and fumarase but not malate dehydrogenase was also given when the enzymes were heated in the presence of their substrates. These results are discussed in light of the generalized theories of facultative thermophily which have been proposed.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.     

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10(-6)M for aconitase, 10(-4)M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10(-3)M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10(-3)M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The Ki values for the purified enzyme in the presence of 5 microM alloxan were 0.22 microM with citrate, 4.0 microM with cis-aconitate and 0.62 microM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.