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We present the results of genetical and biochemical investigations of yeast mutants which presumably are deficient in different steps of the Krebs cycle. The investigation of the activities of the Krebs cycle enzymes suggests that we have isolated two types of mutants: structural and regulatory.
Krebs cycle enzyme activity in Bacillus subtilis was examined under aerobic and anaerobic conditions. Citrate synthase and aconitase activities in cells grown anaerobically in the presence of nitrate were reduced by as much as 10- and 30-fold, respectively, from levels observed under aerobic culture conditions. The maximum level of isocitrate dehydrogenase activity during anaerobic growth was only twofold lower than that in aerobic cultures. These reductions in activity under conditions of anaerobiosis were found to be primarily the result of reduced Krebs cycle gene transcription. This repression was not dependent on either the fnr or resDE gene products, which have been shown to regulate expression of other B. subtilis genes in response to anaerobic conditions. Additionally, catabolite control proteins CcpA and CcpB were not responsible for the repression. A dyad symmetry element located between positions -73 and -59 relative to the transcription start site of the aconitase gene (citB) promoter was previously shown to be a target of catabolite repression and the binding site for a putative negative regulator during aerobic growth. The deletion of the upstream arm of the dyad symmetry region abolished the citB repression observed during anaerobic growth. Furthermore, neither citZ or citB was repressed in an anaerobically grown citB mutant, an effect that was very likely the result of citrate accumulation. These results suggest that catabolite repression and anaerobic repression of citZ and citB are regulated by a common mechanism that does not involve CcpA, CcpB, Fnr, or ResDE.
A practical method using matrix operations is proposed for studying the isotopic transformation of glutamate, or any other metabolite isotopomers, in the Krebs cycle. Two mathematical models were constructed for evaluating the Krebs cycle flux where the enrichment of [2-13C]acetyl-CoA is not 100% and the total glutamate concentration remains constant or varies during incubation. A comparative study of [1-13C]glucose metabolism was subsequently carried out using Saccharomyces cerevisiae cells from two different strains (ATCC-9763 and NCYC-239) by 13C-NMR spectroscopy and biochemical techniques. The results show that there are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2-oxoglutarate dehydrogenase and hence the flux in the Krebs cycle is negligible. With [1-13C]glucose as a carbon source, the 13C-NMR spectra of glutamate exhibit the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1-13C]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The second cell type is represented by NCYC-239. The C2 and C3 areas are equivalent and smaller than the C4 resonance. The results suggest that labeled metabolites enter the Krebs cycle only at the citrate level via acetyl-CoA, 2-oxoglutarate dehydrogenase is present but pyruvate carboxylase is virtually absent or inactivated. When both are incubated with glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchange with the Krebs cycle in NCYC-239 cells is about 2.6% and the reduction in glutamate concentration is about 0.5%/min. Using our model, with a variable glutamate pool size, good agreement between the theoretical and experimental data is obtained.
Krebs cycle intermediates such as succinate, citrate, and alpha-ketoglutarate are transferred across plasma membranes of cells by secondary active transporters that couple the downhill movement of sodium to the concentrative uptake of substrate. Several transporters have been identified in isolated membrane vesicles and cells based on their functional properties, suggesting the existence of at least three or more Na+/dicarboxylate cotransporter proteins in a given species. Recently, several cDNAs, called NaDC-1, coding for the low-affinity Na+/dicarboxylate cotransporters have been isolated from rabbit, human, and rat kidney. The Na+/dicarboxylate cotransporters are part of a distinct gene family that includes the renal and intestinal Na+/sulfate cotransporters. Other members of this family include a Na(+)- and Li(+)-dependent dicarboxylate transporter from Xenopus intestine and a putative Na+/dicarboxylate cotransporter from rat intestine. The current model of secondary structure in NaDC-1 contains 11 transmembrane domains and an extracellular N-glycosylated carboxy terminus.
We have applied an algorithmic methodology which provably decomposes any complex network into a complete family of principal subcircuits to study the minimal circuits that describe the Krebs cycle. Every operational behavior that the network is capable of exhibiting can be represented by some combination of these principal subcircuits and this computational decomposition is linearly efficient. We have developed a computational model that can be applied to biochemical reaction systems which accurately renders pathways of such reactions via directed hypergraphs (Petri nets). We have applied the model to the citric acid cycle (Krebs cycle). The Krebs cycle, which oxidizes the acetyl group of acetyl CoA to CO(2) and reduces NAD and FAD to NADH and FADH(2), is a complex interacting set of nine subreaction networks. The Krebs cycle was selected because of its familiarity to the biological community and because it exhibits enough complexity to be interesting in order to introduce this novel analytic approach. This study validates the algorithmic methodology for the identification of significant biochemical signaling subcircuits, based solely upon the mathematical model and not upon prior biological knowledge. The utility of the algebraic-combinatorial model for identifying the complete set of biochemical subcircuits as a data set is demonstrated for this important metabolic process.
Among the Krebs cycle components, just citrate enhances the fluorescence of a new bi(brachial) lariat aza-crown containing appended naphthalene fluorophores.
The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible. It liberates CO2 and generates NADH whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO2 assimilation. In 1966, our laboratory described a cyclic pathway for CO2 assimilation (Evans, Buchanan and Arnon 1966) that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO2 by a pathway different from Calvin's reductive pentose phosphate cycle (Calvin 1962) and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle. Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO2 assimilation pathway in Chlorobium thiosulfatophilum (Evans et al. 1966) (now known as Chlorobium limicola forma thiosulfatophilum). Chlorobium is a photosynthetic green sulfur bacterium that grows anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO2 as an obligatory carbon source. In the ensuing years, the new cycle was viewed with skepticism. Not only was it in conflict with the prevailing doctrine that the 'one important property ... shared by all (our emphasis) autotrophic species is the assimilation of CO2 via the Calvin cycle' (McFadden 1973) but also some of its experimental underpinnings were challenged. It is only now that in the words of one of its early skeptics (Tabita 1988) 'a long and tortuous controversy' has ended with general acceptance of the reductive carboxylic acid cycle as a photosynthetic CO2 assimilation pathway distinct from the pentose cycle. (Henceforth, to minimize repetitiveness, the reductive pentose phosphate cycle will often be referred to as the pentose cycle and the reductive carboxylic acid cycle as the carboxylic acid cycle.) Aside from photosynthetic pathways which are the focus of this article, CO2 assimilation is also known to sustain autotrophic growth via the acetyl-CoA pathway (Wood et al. 1986). Our aim here is to discuss (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO2 assimilation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.
Krebs cycle disorders constitute a group of rare human diseases which present an amazing complexity considering our current knowledge on the Krebs cycle function and biogenesis. Acting as a turntable of cell metabolism, it is ubiquitously distributed in the organism and its enzyme components encoded by supposedly typical house-keeping genes. However, the investigation of patients presenting specific defects of Krebs cycle enzymes, resulting from deleterious mutations of the considered genes, leads to reconsider this simple envision by revealing organ-specific impairments, mostly affecting neuromuscular system. This often leaves aside organs the metabolism of which strongly depends on mitochondrial energy metabolism as well, such as heart, kidney or liver. Additionally, in some patients, a complex pattern of tissue-specific enzyme defect was also observed. The lack of functional additional copies of Krebs cycle genes suggests that the complex expression pattern should be ascribed to tissue-specific regulations of transcriptional and/or translational activities, together with a variable cell adaptability to Krebs cycle functional defects.
The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.
To quantify intrahepatic Krebs cycle metabolism, phenyl acetate, excreted in urine as a glutamine conjugate, was given to healthy subjects infused with [3-14C]lactate. They were studied after 60 h of fasting and when given glucose after an overnight fast. Distributions of 14C in glutamate from urinary phenylacetylglutamine and blood glucose were determined. Corrections to the distributions because of the fixation of 14CO2 formed from the [3-14C]lactate were determined by administering [14C]bicarbonate. Comparisons of distributions in glucose and glutamate support the assumption that the glutamate distributions reflect those in hepatic alpha-ketoglutarate. From the distributions in glutamate, the extent of exchange of labeled with unlabeled carbons and relative flow rates in the cycle in liver were estimated. Dilution of 14C by 12C in the cycle was found in the fasted but not the fed state. In the fasted state, pyruvate carboxylation was estimated to be at least twice the rate of Krebs cycle flux and the rate of pyruvate's decarboxylation less than 1/25 the rate of its carboxylation. In the fed state, the rate of decarboxylation was estimated to be between one-sixth and one-half the rate of carboxylation. The rate of conversion of oxalacetate to fumarate in both states appeared to be greater than 6 times the rate of Krebs cycle flux.
The investigation of Krebs cycle state in kidney homogenates of August rats subjected to oral intoxication with oil solution of yellow phosphorus in a dose of 0.3 mg/kg, has shown that under conditions of balanced nutrition the activity of NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase and accumulation of the substrate fund of the cycle decreased 3.5-fold as compared to the control. The addition of polyunsaturated fatty acids to the ration produced a positive effect on Krebs cycle state: dehydrogenase activity was not significantly changed, accumulation of Krebs cycle substrate was two-fold lower. However, this ration did not completely abolish the toxic action of yellow phosphorus on Krebs cycle.
Objective : Recent studies have proposed that Krebs cycle metabolites may serve as potential biomarkers for prognosis in sepsis. However, whether these metabolites are associated with disease severity and can be applied to improve the effectiveness of current prognosis assessment in sepsis remains unclear and is explored in this study. Methods : This prospective multicenter cohort study was conducted in medical intensive care units (ICUs). From December 2019 to September 2022, consecutive patients admitted to medical ICUs for sepsis were screened and recruited. Plasma samples were obtained for measurements of cytokines and Krebs cycle metabolites, including citrate/isocitrate, cis-aconitate, alpha-ketoglutarate, succinate, fumarate, and malate. Results : In total, 97 patients admitted for sepsis were enrolled in the study. The 28-day mortality rate was 17.5%, and nonsurvivors exhibited significantly increased plasma lactate levels and Sequential Organ Failure Assessment (SOFA) scores. Plasma levels of Krebs cycle metabolites were significantly correlated with both plasma lactate and interleukin-6 levels. Except for citrate/isocitrate, all Krebs cycle metabolites were significantly elevated in patients with acute kidney injury. Multivariate Cox proportional hazard models, adjusted for plasma lactate levels and SOFA scores, revealed that plasma levels of alpha-ketoglutarate (adjusted hazard ratio [HR]: 2.404, P = 0.002), fumarate (adjusted HR: 1.904, P = 0.001) and malate (adjusted HR: 1.327, P = 0.019) were associated with increased risk of 28-day mortality. Conclusions : Study findings indicate that Krebs cycle metabolites, particularly alpha-ketoglutarate, fumarate, and malate, when applied with SOFA score, might enhance prognostic assessment in patients with sepsis.
Oxidation of [2,3-14C]succinate in the intramitochondrial Krebs cycle was used as a probe to investigate the effect of ammonia on protein incorporation and Krebs cycle oxidation of succinate carbons in isolated rat hepatocytes. At low concentrations of ammonium chloride (0.1 to 0.5 mM) a slight increase in 14CO2 formation from [2,3-14C]succinate was observed, however, the stimulatory effect of insulin was significantly reduced. Insulin failed to cause any stimulation of succinate carbons incorporation into hepatocyte protein in the presence of ammonium chloride. Addition of ammonium chloride also depressed the movement of tracer carbons into the gluconeogenesis pathway. The activity of the amphibolic amino acid pool was significantly enhanced by ammonia. The data presented in this paper lend strong support to the Krebs-cycle depletion theory of hepatic coma. They also suggest that reduced mitochondrial Krebs cycle activity caused by increased amphibolic depletion of substrates results in loss of insulin sensitivity in ammonia toxicity.
The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5-3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), alpha-ketoglutaric acid (alpha-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with alpha-KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with alpha-KGA than through reductive amination of alpha-KGA. The experimental data proved that intact bacteroids possess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon. The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N2-fixation, and sucrose available from leaves.
Purposes of this study were 1) to estimate in humans, using 14C-labeled propionate, the rate of hepatic gluconeogenesis relative to the rate of Krebs cycle flux; 2) to compare those rates with estimates previously made using [3-14C]lactate and [2-14C]acetate; 3) to determine if the amount of ATP required for that rate of gluconeogenesis could be generated in liver, calculated from that rate of Krebs cycle flux and splanchnic balance measurements, previously made, and 4) to test whether hepatic succinyl-CoA is channeled during its metabolism through the Krebs cycle. [2-14C]propionate, [3-14C]-propionate, and [2,3-14C]succinate were given along with phenyl acetate to normal subjects, fasted 60 h. Distributions of 14C were determined in the carbons of blood glucose and of glutamate from excreted phenylacetylglutamine. Corrections to the distributions for 14CO2 fixation were made from the specific activities of urinary urea and the specific activities in glucose, glutamate, and urea previously found on administering [14C]-bicarbonate. Uncertainties in the corrections and in the contributions of pyruvate and Cori cyclings limit the quantitations. The rate of gluconeogenesis appears to be two or more times the rate of Krebs cycle flux and pyruvate's decarboxylation to acetyl-CoA, metabolized in the cycle, less than one-twenty-fifth the rate of its decarboxylation. Such estimates were previously made using [3-14C]lactate. The findings support the use of phenyl acetate to sample hepatic alpha-ketoglutarate. Ratios of specific activities of glucose to glutamate and glucose to urinary urea and expired CO2 indicate succinate's extensive metabolism when presented in trace amounts to liver. Utilizations of the labeled compounds by liver relative to other tissues were in the order succinate = lactate > propionate > acetate. ATP required for gluconeogenesis and urea formation was approximately 40% of the amount of ATP generated in liver. There was no channeling of succinyl-CoA in the Krebs cycle in the hepatic mitochondria.
Krebs cycle enzyme activities and levels of five metabolites were determined from livers of old mice (30 months) maintained either on control or on long-term caloric restriction (CR) diets (28 months). In CR mice, the cycle was divided into two major blocks, the first containing citrate synthase, aconitase and NAD-dependent isocitrate dehydrogenase which showed decreased activities, while the second block, containing the remaining enzymes, displayed increased activity (except for fumarase, which was unchanged). CR also resulted in decreased levels of citrate, glutamate and alpha-ketoglutarate, increased levels of malate, and unchanged levels of aspartate. The alpha-ketoglutarate/glutamate and malate/alpha-ketoglutarate ratios were higher in CR, in parallel with previously reported increases with CR in pyruvate carboxylase activity and glucagon levels, respectively. The results indicate that long-term CR induces a differential regulation of Krebs cycle in old mice and this regulation may be the result of changes in gene expression levels, as well as a complex interplay between enzymes, hormones and other effectors. Truncation of Krebs cycle by CR may be an important adaptation to utilize available substrates for the gluconeogenesis necessary to sustain glycolytic tissues, such as brain.
Uninvolved gastric mucosa from duodenal ulcer, gastric ulcer, and gastric cancer patients was incubated with [1-14C]glucose and [6-14C]glucose in order to assess the relative contributions of the pentose phosphate pathway and Krebs cycle to glucose metabolism. [14C]Glucose counts retained by the tissue, glycolysis, and pyruvate formation were also measured. Tumor tissue from the cancer patients was included in the study. Less than 1.2% of the glucose entering the tissues was metabolized via the pentose phosphate pathway; suggesting that this pathway plays a minor role in energy production from glucose. The major determinant of energy production was the Krebs cycle. Its contribution to glucose metabolism was greatest in the body mucosa of duodenal ulcer patients, less in the uninvolved body mucosa of gastric ulcer patients, and lower still in the corresponding body mucosa of gastric cancer patients. The low levels of Krebs cycle activity seen in the latter tissue resembled those of uninvolved antral mucosa. The smallest Krebs cycle contribution was seen in tumor tissue. [14C]Glucose counts retained by the tissue and glycolysis both tended to vary inversely with Krebs cycle activity among the tissues studied. Thus, both were small in the body mucosa of noncancer patients and somewhat larger in the body mucosa of cancer patients, in uninvolved antral mucosa and in tumor tissue.
A variant Chinese hamster cell line has been isolated from a mutagenized population that has a markedly reduced ability to oxidize a variety of substrates via the Krebs cycle. The production of 14CO2 from 14C-labeled compounds was measured using pyruvate, acetate, beta-hydroxybutyrate, palmitate and glutamate, and in all cases it was neglibible in the mutant. In contrast to this, significant amounts of 14CO2 were produced from 14C-aspartate and 14C-succinate which suggest that some reactions of the Krebs cycle can take place and this conclusion is supported by tracer experiments with labeled compounds. The rate of respiration measured with a Clark oxygen electrode in the mutant was compared to several normal Chinese hamster cell lines and was found to be only 8%. Mitochondria appear to be present in normal numbers and with only minor differences in morphology. The measurement of difference spectra between oxidized and reduced states permits us to conclude that the cytochromes are all present and functional. These results lead us to believe that there may be a defect in the Krebs cycle between alpha-ketoglutarate and succinate. Alternatively a defect in a structural component of the mitochondria or in the electron-transport chain itself may be causing pleiotropic effects in the Krebs cycle and respiration.