Chapter X Assays of Enzymes of the Tricarboxylic Acid and Glyoxylate Cycles

Reeves, Henry C.; Rabin, Robert; Wegener, Warner S.; Ajl, Samuel J.

doi:10.1016/s0580-9517(08)70582-8

Cited by 53 publications

(47 citation statements)

References 73 publications

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“…Crude cell extracts for in vitro enzyme assays were prepared from pellets obtained from 45-ml culture aliquots. The pellets were resuspended in 4 ml of 0.9% (wt/vol) NaCl-10 mM MgSO 4 , passed three times through a French pressure cell (1.2 ϫ 10 8 Pa), and centrifuged at 11,000 ϫ g. Isocitrate dehydrogenase (34) and 2-oxoglutarate dehydrogenase (53) were assayed at 25°C by using coupled optical tests with NADP ϩ and NAD ϩ , respectively, as the acceptors. Succinate dehydrogenase activity was assayed by determining the succinate-dependent reduction of 2,6-dichlorophenol-indophenol at 25°C (43).…”

Section: Methodsmentioning

confidence: 99%

Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

2005

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Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.Metabolic networks consist of hundreds of metabolites that are interconnected through a large number of biochemical and regulatory reactions. In principle, metabolites could flow through various reactions, but only few specific pathways are used in reality (20). This distribution of molecular fluxes is regulated by multiple mechanisms at several levels that include gene expression, posttranscriptional control, enzyme kinetics, and allosteric control. While transcriptional regulation is generally considered the main mode of regulation in bacteria, the extent to which it actually controls the distribution of metabolic fluxes through different pathways is mostly unknown. Based on metabolism-wide comparisons of metabolic flux and gene expression during growth on different substrates, the flux through some central metabolic pathways was found to correlate, at least qualitatively, with the expression level, but in many cases there was no apparent correlation (12,24,41,42). In parasitic protists, the glycolytic flux was demonstrated to be rarely completely controlled at the transcriptional level and mostly not even largely controlled at this level (65).Transcriptional regulation itself involves a complex network of global and specific regulators, in which global regulators exhibit pleiotropic phenotypes and regulate several operons that belong to different functional groups (26). In Escherichia coli, only seven global regulators (ArcA, Crp, Fis, Fnr, Ihf, Lrp, and NarL) directly modulate the expression of about one-half of all genes (39). The following three regulators have specific metabolic functions that involve altering expression of genes that are involved in central carbon m...

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Section: Methodsmentioning

confidence: 99%

Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

2005

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“…Membrane and soluble fractions for enzymic assays were prepared from batch or chemostat cultures under anaerobic conditions as described by Kaufmann & Lovley (2001) (Reeves et al, 1971). Pyruvate-ferredoxin oxidoreductase and 2-oxoglutarate-ferredoxin oxidoreductase activity were determined by monitoring pyruvate-or oxoglutarate-dependent reduction of benzyl viologen at 578 nM (e58.6 mM 21 cm 21 ) in a modified form of the assay buffer used by Brandis-Heep et al (1983), which consisted of 100 mM Tricine/NaOH (pH 8.5), 0.05 mM coenzyme A, 0.1 mM thiamine pyrophosphate, 5 mM MgCl 2 , 5 mM b-mercaptoethanol, 1 mM benzyl viologen and either 10 mM 2-oxoglutarate or 10 mM pyruvate.…”

Section: Methodsmentioning

confidence: 99%

Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction

et al. 2007

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A soluble ferric reductase, SfrAB, which catalysed the NADPH-dependent reduction of chelated Fe(III), was previously purified from the dissimilatory Fe(III)-reducing micro-organism Geobacter sulfurreducens, suggesting that reduction of chelated forms of Fe(III) might be cytoplasmic. However, metabolically active spheroplast suspensions could not catalyse acetate-dependent Fe(III) citrate reduction, indicating that periplasmic and/or outer-membrane components were required for Fe(III) citrate reduction. Furthermore, phenotypic analysis of an SfrAB knockout mutant suggested that SfrAB was involved in acetate metabolism rather than respiration-linked Fe(III) reduction. The mutant could not grow via the reduction of either Fe(III) citrate or fumarate when acetate was the electron donor but could grow with either acceptor if either hydrogen or formate served as the electron donor. Following prolonged incubation in acetate : fumarate medium in the absence of hydrogen and formate, an 'acetate-adapted' SfrAB-null strain was isolated that was capable of growth on acetate : fumarate medium but not acetate : Fe(III) citrate medium. Comparison of gene expression in this strain with that of the wild-type revealed upregulation of a potential NADPH-dependent ferredoxin oxidoreductase as well as genes involved in energy generation and amino acid uptake, suggesting that NADPH homeostasis and the tricarboxylic acid (TCA) cycle were perturbed in the 'acetate-adapted' SfrAB-null strain. Membrane and soluble fractions prepared from the 'acetate-adapted' strain were depleted of NADPH-dependent Fe(III), viologen and quinone reductase activities. These results indicate that cytoplasmic, respiration-linked reduction of Fe(III) by SfrAB in vivo is unlikely and suggest that deleting SfrAB may interfere with growth via acetate oxidation by interfering with NADP regeneration.

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“…Following centrifugation at 30000 g for 20 min, the supernatant was used for enzyme assay. Citrate synthase (EC 4.1.3.7), 2-oxoglutarate dehydrogenase (EC 1.2.4.2), isocitrate dehydrogenase (EC 1.1.1.41) and succinyl-CoA synthetase (EC 6.2.1.5) were assayed according to Reeves et al (1971). Malate dehydrogenase (EC 1.1.1.37) was assayed by the technique of Saroso et al (1986).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

et al. 1997

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Reading RG6 6AJ, UKMutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to s u d and suct), which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex ( s u d B ) . Mutation of either SUCC or SUCD prevents expression of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes mdh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of s u d or suct), the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino acid uptake via the Aap of R. leguminosamm was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC). Mutants of succinyl-CoA synthetase (SUCD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and reductant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.

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Chapter X Assays of Enzymes of the Tricarboxylic Acid and Glyoxylate Cycles

Cited by 53 publications

References 73 publications

Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

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