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PubMed1976

Mutants of Escherichia coli K12 unable to use fumarate as an anaerobic electron acceptor.

Mutants of Escherichia coli K12 strain WGAS-GF+/LF+ were selected for their inability to use fumarate as terminal electron acceptor for supporting growth on glycerol or lactate in an atmosphere of H2 plus 5% CO2. Eighty-three mutants were grouped into seven different categories according to their ability to grow on different media and their ability to produce gas during glucose fermentation. Enzymological and genetic studies indicated that the major class (type I), representing nearly 70% of the isolates, lacked fumarate reductase and corresponded to the frdA mutants studied previously (Spencer & Guest, 1973, 1974). Members of a second class (type II) were phenotypically similar to men mutants, blocked in menaquinone biosynthesis. They differed from menA mutants in having lesions in the 44 to 51 min region of the chromosome rather than at 87 min. It was concluded that fumarate reductase and menaquinone are essential for anaerobic growth when fumarate serves as electron acceptor but not when nitrate performs this function. Fumarate reductase and menaquinone are also essential for H2-dependent growth on fumarate. Type III mutants, originally frdB, were designated fnr because they were defective in fumarate and nitrate reduction and impaired in their ability to produce gas. The fnr gene was located at 28-5 min by its cotransducibility with pyrF (5-7 to 9-2%) and trpA (2-7 to 5-7%) and the gene order fnr-qmeA-pyrF-trpA was established. It was not possible to assign specific metabolic lesions to the fnr mutants nor to the remaining classes, which all exhibited pleiotropic phenotypes. Nevertheless, the results demonstrate that functional or organizational relationships exist between the fumarate reductase system, nitrate reduction and hydrogen production.

Aldehyde Oxidoreductases↗
PubMed1977

Hydrogenase activity and the H2-fumarate electron transport system in Bacteroides fragilis.

Hydrogenase activity and the H(2)-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H(2). Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H(2) was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H(2), but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H(2) when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H(2) to fumarate in B. fragilis is proposed.

Acriflavine↗
PubMed1999

Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation.

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.

Actinobacillus↗
PubMed1989

An unusual thiol-driven fumarate reductase in Methanobacterium with the production of the heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine-O3-phosphate.

An unusual fumarate reductase was purified from cell extracts of Methanobacterium thermoautotrophicum and partially characterized. Two coenzymes previously isolated from cell extracts, 2-mercaptoethane-sulfonic acid (HS-CoM) and N-(7-mercaptoheptanoyl)threonine-O3-phosphate (HS-HTP), were established as direct electron donors for fumarate reductase. By measuring the consumption of free thiol, we determined that fumarate reductase catalyzed the oxidation of HS-CoM and HS-HTP; by the direct measurement of succinate and the heterodisulfide of HS-CoM and HS-HTP (CoM-S-S-HTP), we established that these compounds were products of the fumarate reductase reaction. A number of thiol-containing compounds did not function as substrates for fumarate reductase, but this enzyme had high specific activity when HS-CoM and HS-HTP were used as electron donors. HS-CoM and HS-HTP were quantitatively oxidized by the fumarate reductase reaction, and results indicated that this reaction was irreversible. Additionally, by measuring formylmethanofuran, we demonstrated that the addition of fumarate to cell extracts activated CO2 fixation for the formation of formylmethanofuran. Results indicated that this activation resulted from the production of CoM-S-S-HTP (a compound known to be involved in the activation of formylmethanofuran synthesis) by the fumarate reductase reaction.

Disulfides↗
PubMed1993

Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction.

A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.

Animals↗
PubMed2002

Effects of bisoprolol fumarate on left ventricular size, function, and exercise capacity in patients with heart failure: analysis with magnetic resonance myocardial tagging.

BACKGROUND: Recent data suggest that beta-blockers can be beneficial in subgroups of patients with chronic heart failure (CHF). For metoprolol and carvedilol, an increase in ejection fraction has been shown and favorable effects on the myocardial remodeling process have been reported in some studies. We examined the effects of bisoprolol fumarate on exercise capacity and left ventricular volume with magnetic resonance imaging (MRI) and applied a novel high-resolution MRI tagging technique to determine myocardial rotation and relaxation velocity. METHODS: Twenty-eight patients (mean age, 57 +/- 11 years; mean ejection fraction, 26 +/- 6%) were randomized to bisoprolol fumarate (n = 13) or to placebo therapy (n = 15). The dosage of the drugs was titrated to match that of the the Cardiac Insufficiency Bisoprolol Study protocol. Hemodynamic and gas exchange responses to exercise, MRI measurements of left ventricular end-systolic and end-diastolic volumes and ejection fraction, and left ventricular rotation and relaxation velocities were measured before the administration of the drug and 6 and 12 months later. RESULTS: After 1 year, heart rate was reduced in the bisoprolol fumarate group both at rest (81 +/- 12 before therapy versus 61 +/- 11 after therapy; P <.01) and peak exercise (144 +/- 20 before therapy versus 127 +/- 17 after therapy; P <.01), which indicated a reduction in sympathetic drive. No differences were observed in heart rate responses in the placebo group. No differences were observed within or between groups in peak oxygen uptake, although work rate achieved was higher (117.9 +/- 36 watts versus 146.1 +/- 33 watts; P <.05) and exercise time tended to be higher (9.1 +/- 1.7 minutes versus 11.4 +/- 2.8 minutes; P =.06) in the bisoprolol fumarate group. A trend for a reduction in left ventricular end-diastolic volume (-54 mL) and left ventricular end-systolic volume (-62 mL) in the bisoprolol fumarate group occurred after 1 year. Ejection fraction was higher in the bisoprolol fumarate group (25.0 +/- 7 versus 36.2 +/- 9%; P <.05), and the placebo group remained unchanged. Most changes in volume and ejection fraction occurred during the latter 6 months of treatment. With myocardial tagging, insignificant reductions in left ventricular rotation velocity were observed in both groups, whereas relaxation velocity was reduced only after bisoprolol fumarate therapy (by 39%; P <.05). CONCLUSION: One year of bisoprolol fumarate therapy resulted in an improvement in exercise capacity, showed trends for reductions in end-diastolic and end-systolic volumes, increased ejection fraction, and significantly reduced relaxation velocity. Although these results generally confirm the beneficial effects of beta-blockade in patients with chronic heart failure, they show differential effects on systolic and diastolic function.

Adrenergic beta-Antagonists↗
PubMed2003

Iron absorption from ferrous fumarate in adult women is influenced by ascorbic acid but not by Na2EDTA.

Ascorbic acid and Na2EDTA enhance Fe absorption from the water-soluble Fe compound FeSO4 but their effect on poorly water-soluble Fe compounds such as ferrous fumarate is less well established. In the present study, the effects of ascorbic acid and Na2EDTA on Fe absorption from ferrous fumarate were evaluated in adult women (ten women/study) from the erythrocyte incorporation of Fe stable isotopes ((57)Fe or (58)Fe) 14 d after administration. Two separate studies were made with test meals of Fe-fortified infant cereal (5 mg Fe/meal). Data were evaluated by paired t tests and the results are presented as geometric means. In study 1a, the comparison between Fe absorption from ferrous fumarate- and FeSO4-fortified cereal showed that adult women absorb Fe as well from ferrous fumarate as from FeSO4 (3.0 and 3.1 % respectively, P=0.85). After addition of Na2EDTA (Na2EDTA:fortification Fe molar ratio of 1:1), Fe absorption from FeSO4 was significantly higher than from ferrous fumarate (5.3 v. 3.3 % respectively, P<0.01; study 1b). In study 2, Fe absorption was compared from ferrous fumarate-fortified meals with and without ascorbic acid added at a 4:1 molar ratio (relative to fortification Fe) and the results showed that ascorbic acid increased Fe absorption from ferrous fumarate significantly (6.3 v. 10.4 %, P=0.02). The results of the present studies show that Fe absorption from ferrous fumarate is enhanced by ascorbic acid but not by Na2EDTA, thus emphasising that not all findings from Fe absorption studies made with FeSO4 can be extrapolated to Fe compounds with different solubility properties.

Adult↗
PubMed1994

Fumarate reductase of Helicobacter pylori--an immunogenic protein.

An immunogenic protein with an apparent mol. wt of 80 kDa that was recognised by 55% of sera from patients infected with Helicobacter pylori in Western blots was found in butanol extracts of H. pylori membranes. The N-terminal amino-acid sequence of the 80-kDa protein showed 80% identity with the N-terminal sequence of subunit A of the fumarate reductase of Wolinella succinogenes, suggesting the existence of a fumarate reductase in H. pylori. The membrane fraction of H. pylori catalysed succinate oxidation with methylene blue at a specific enzyme activity of 0.06 U/mg of protein. The enzyme was purified by Triton X100 extraction followed by ion-exchange chromatography. The purified enzyme contained an 80-kDa protein which was recognised by rabbit serum raised against subunit A of fumarate reductase of W. succinogenes. A second protein band with a mol. wt of 31 kDa was recognised by rabbit serum raised against subunit B of fumarate reductase of W. succinogenes. Two-dimensional gel electrophoresis demonstrated that the 80- and 31-kDa proteins were subunits of one protein complex. These results indicate that H. pylori contains an enzyme that is very similar to W. succinogenes fumarate reductase. The 80-kDa subunit was recognised in sonicates of all 32 H. pylori strains tested by rabbit antibodies raised against subunit A of fumarate reductase of W. succinogenes, indicating that fumarate reductase is a common protein in H. pylori. The fumarate reductase of H. pylori might enable the bacterium to perform anaerobic respiration in a similar fashion to other anaerobic or facultative bacteria.

Amino Acid Sequence↗
PubMed1962

Production of Fumaric Acid in 20-Liter Fermentors.

The conditions necessary for the production of fumaric acid in 20-liter fermentors by fermentation of glucose with Rhizopus arrhizus strain NRRL 2582 were determined. Continuous neutralization of fumaric acid was necessary for optimal yields. Yields of the calcium salt were in excess of 65 g of fumaric acid from 100 g of sugar consumed during fermentation of sugar concentrations of 10 to 16%. Conditions established for calcium fumarate production include a simple mineral salts medium, 0.5 v:v:min aeration rate, 300 rev/min agitation rate in a baffled tank, 33 C incubation temperature, CaCO(3) to neutralize the acid formed, and a 4 to 5% (v/v) vegetative inoculum. A suitable procedure and medium for the preparation of a vigorous vegetative inoculum were established. The tendency for calcium fumarate fermentations to foam excessively was controlled with a proper antifoam agent added prior to sterilization of the medium and again at daily intervals during fermentation. The production of soluble sodium or potassium fumarates was inhibited when the concentration of fumarates reached 3.5 to 4.0%. No means of overcoming this inhibition was found. Starches and certain other grain-derived carbohydrates were fermented to form calcium fumarate in flask experiments with approximately the same efficiency as was glucose.

Journal Article↗
PubMed1995

Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain.

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.

Aerobiosis↗
PubMed2003

Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters.

Psoriasis is a chronic inflammatory skin disease. Its treatment is based on the inhibition of proliferation of epidermal cells and interference in the inflammatory process. A new systemic antipsoriasis drug, which consists of dimethylfumarate and ethylhydrogenfumarate in the form of their calcium, magnesium and zinc salts has been introduced in Europe with successful results. In the present study, a homologous series of mono- and diesters of fumaric acid has been studied with respect to the sites and kinetics of presystemic ester degradation using pancreas extract, intestinal perfusate, intestinal homogenate and liver S9 fraction. In addition, intestinal permeability has been determined using isolated intestinal mucosa as well as Caco-2 cell monolayers, in order to obtain estimates of the fraction of the dose absorbed for these compounds. Relationships between the physicochemical properties of the fumaric acid esters and their biological responses were investigated. The uncharged diester dimethylfumarate displayed a high presystemic metabolic lability in all metabolism models. It also showed the highest permeability in the Caco-2 cell model. However, in permeation experiments with intestinal mucosa in Ussing-type chambers, no undegraded DMF was found on the receiver side, indicating complete metabolism in the intestinal tissue. The intestinal permeability of the monoesters methyl hydrogen fumarate, ethyl hydrogen fumarate, n-propylhydrogen fumarate and n-pentyl hydrogen fumarate increased with an increase in their lipophilicity, however, their presystemic metabolism rates likewise increased with increasing ester chain length. It is concluded that for fumarates, an increase in intestinal permeability of the more lipophilic derivatives is counterbalanced by an increase in first-pass extraction.

Animals↗
PubMed1978

[In vitro incubation of lenses. Model for testing substrate utilization of substances of the energy metabolism, demonstrated with bencyclane-hydrogen-fumarate (author's transl)].

When bovine lens homogenate was treated with bencyclane-hydrogen-fumarate, the carbohydrate metabolism was activated. This may chiefly be due to the fumarate part of the substance. A 24 H In vitro incubation of whole bovine lenses in TC-199 with and without bencyclane-hydrogen-fumarate did not show the above effect. On the model of former investigations by J.E. Harris et al. we modified the test procedure by selecting the medium and the time of incubation so that the endogenous carbohydrates of the lens were consumed, thus creating new metabolic balances. This metabolic condition allows investigations intended to activate metabolic processes and to restore the steady state of metabolic parameters. We investigated the effect of bencyclane-hydrogen-fumarate using the same method and found that given certain conditions the lens recovers when incubated for 2 h in TC-199 (containing 1 g glucose/1) with addition of a 10(-4) M solution of bencyclane-hydrogen-fumarate. The ATP-content of these lenses in particular gives proof of this result. As already observed in former investigations on homogenates, this effect is probably due to metabolization of the fumarate part of the bencyclane-hydrogen-fumarate by the citric acid cycle. The method used explains the differences observed when using lens homogenates or whole lenses under the same experimental conditions.

Adenine Nucleotides↗
PubMed2004

Quantifying the effects of fumarate on in situ reductive dechlorination rates.

In situ methods are needed to evaluate the effectiveness of chemical amendments at enhancing reductive dechlorination rates in groundwater that is contaminated with the priority pollutant, trichloroethene (TCE). In this communication, a method that utilizes single-well, "push-pull" tests to quantify the effects of chemical amendments on in situ reductive dechlorination rates is presented and demonstrated. Five push-pull tests were conducted in each of five monitoring wells located in a TCE-contaminated aquifer at the site of a former chemical manufacturing facility. Rates for the reductive dechlorination of the fluorinated TCE-surrogate, trichlorofluoroethene (TCFE), were measured before (test 1) and after (test 5) three successive additions (tests 2-4) of fumarate. Fumarate was selected to stimulate the growth and activity of indigenous microorganisms with the metabolic capability to reduce TCFE and TCE. In three wells, first-order rate constants for the reductive dechlorination of TCFE increased by 8.2-92 times following fumarate additions. In two wells, reductive dechlorination of TCFE was observed after fumarate additions but not before. The transformation behavior of fumarate was also monitored following each fumarate addition. Correlations between the reductive dechlorination of TCFE and the reduction of fumarate to succinate were observed, indicating that these reactions were supported by similar biogeochemical conditions at this site.

Biodegradation, Environmental↗
PubMed2000

Catalysis in fumarate reductase.

In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.

Binding Sites↗
PubMed1997

Schistosoma mansoni sporocysts contain rhodoquinone and produce succinate by fumarate reduction.

Although schistosomes were thought to be one of the few parasitic helminths that do not produce succinate via fumarate reduction, it was recently demonstrated that sporocysts of Schistosoma mansoni produce, under certain conditions, succinate in addition to lactate. This succinate production was only observed when the respiratory chain activity of the sporocysts was inhibited, which suggested that succinate is produced by fumarate reduction. In this report the presence of essential components for fumarate reduction was investigated in various stages of S. mansoni and it was shown that, in contrast to adults, sporocysts contained a substantial amount of rhodoquinone which is essential for efficient fumarate reduction in eukaryotes. This rhodoquinone was not made by modification of ubiquinone obtained from the host, but was synthesized de novo. Furthermore, it was shown that complex II of the electron-transport chain in schistosomes has the kinetic properties of a dedicated fumarate reductase instead of those of a succinate dehydrogenase. The presence of such an enzyme, together with the substantial amounts of rhodoquinone, shows that in S. mansoni sporocysts succinate is produced via fumarate reduction. Therefore, the energy metabolism of schistosomes does not differ in principle from most other parasitic helminths, which are known to rely heavily on fumarate reduction.

Animals↗
PubMed2003

Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites.

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.

Allosteric Site↗
PubMed1996

Reduction of dopamine beta-monooxygenase. A unified model for apparent negative cooperativity and fumarate activation.

The interactions of reductants with dopamine beta-monooxygenase (DbetaM) were examined using two novel classes of reductants. The steady-state kinetics of the previously characterized DbetaM reductant, N,N-dimethyl-1,4-p-phenylenediamine (DMPD), were parallel to the ascorbic acid-supported reaction with respect to pH dependence and fumarate activation. DMPD also displayed pH and fumarate-dependent apparent negative cooperativity demonstrating that the previously reported cooperative behavior of DbetaM toward the reductant is not unique to ascorbic acid. The 6-OH phenyl and alkylphenyl-substituted ascorbic acid derivatives were more efficient reductants for the enzyme than ascorbic acid. Kinetic studies suggested that these derivatives behave as pseudo bisubstrates with respect to ascorbic acid and the amine substrate. The lack of apparent cooperative behavior with these derivatives suggests that this behavior of DbetaM is not common for all the reductants. Based on these findings and additional kinetic evidence, the proposal that the apparent negative cooperativity in the interaction of ascorbic acid with DbetaM was due to the presence of a distinct allosteric regulatory site has been ruled out. In contrast to previous models, where fumarate was proposed to interact with a distinct anion binding site, the effect of fumarate on the steady-state kinetics of these novel reductants suggests that fumarate and the reductant may interact with the same site of the enzyme. In accordance with these observations and mathematical analysis of the experimental data, a unified model for the apparent negative cooperativity and fumarate activation of DbetaM in which both fumarate and the reductant interact with the same site of all forms of the enzyme with varying affinities under steady-state turnover conditions has been proposed.

Anticarcinogenic Agents↗
PubMed2006

Fumarate is an essential intermediary metabolite produced by the procyclic Trypanosoma brucei.

The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200).

Animals↗