Mechanisms of desorption and adsorption of liver cytosolic fumarase to cellular membranous components.

The cytosolic fumarase [EC 4.2.1.2[ of rat liver was bound, after dialysis, to the microsomal membrane in vitro. Binding of the enzyme was dependent on pH, and was facilitated in the pH range below 7.5. The binding reaction was completely inhibited by 0.5 mM fumarate, aurintricarboxylate or colchicine. The bound fumarase was released from the membrane by the substrates, isocitrate, citrate or 2,3-diphosphoglycerate at low concentrations. Desorption of the enzyme by metabolites was also dependent on pH, and was more rapid in the alkaline pH range. The enzyme desorption curves were sigmoidal, and kinetic studies suggested a biphasic cooperative mechanism for the action of the metabolites. The apparent desorption constants (concentrations necessary for 50% desorption of the enzyme) estimated at pH 7.3 for isocitrate, 2,3-diphosphoglycerate, L-malate, oxalacetate, fumarate, citrate, succinate, and KCl were 0.073, 0.074, 0.22, 0.39, 0.56, 2.9, and 19 mM, respectively. The bound fumarase showed little enzymatic activity, and its Km and Vmax values were fivefold and 31%, respectively, of those of the free enzyme.     

Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase

The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site.     

5-methyltetrahydrofolate: synthesis and utilization in normal and SV40-transformed BHK-21 cells.

Dodecyl sulfate complexes of two soluble proteins, serum albumin and fumarase, have been “renatured” with large excesses of the nonionic detergent Triton X-100. The resulting complexes, essentially free of dodecyl sulfate, differ in their sedimentation properties relative to the native protein and in their interaction with Triton X-100. Albumin molecules refold to a form binding only very small amounts of Triton and have a sedimentation coefficient similar to that of the non-denatured protein. On the other hand, refolded fumarase molecules have a lower sedimentation coefficient than that of the native enzyme and bind up to 1.06 mg of Triton/mg protein. It is postulated that the fumarase molecule has been turned “inside-out” by the dodecyl sulfate/Triton treatment, and the implications of such large conformational changes for protein transport across membranes are discussed.     

Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54° C. The K _m values for fumarate and l-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe²⁺ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%). Received: 15 September 1995 / Accepted: 13 November 1995     

Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin

Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70 degrees C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58 degrees C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70 degrees C. No acetate kinase activity was observed at 70 degrees C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58 degrees C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70 degrees C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70 degrees C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.     

Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.     

Effect of protein concentration on the molecular weight of delta5-3-ketosteroid isomerase.

The molecular weight of delta-5-3-ketosteroid isomerase from Pseudomonas testosteroni was determined by means of sedimentation equilibrium and exclusion chromatography over a wide range of enzyme concentrations in 0.2 M potassium phosphate buffer, pH 7.0. In addition, the sedimentation constant of the enzyme was determinded over an extended range of concentrations. The enzyme was found to have a molecular weight of 26,000 plus or equal to 1,000, suggesting that it is a dimer of identical or similar 13,400 molecular weight polypeptide chains. In the ultracentrifuge this dimeric species was found to undergo aggregation at enzyme concentrations above 2 mg per ml and dissociation at enzyme concentrations below 0.05 mg per ml. Exclusion chromatography studies indicate that under the conditions of chromatography the oligomeric enzyme is partially dissociated at enzyme concentrations in the range 0.2 to 0.002 mug per ml. These results suggest that under conditions of enzyme assay in 0.2 M potassium phosphate buffer, pH 7.0, isomerase is in a monomeric state of aggregation.     

The denaturation-renaturation of chicken-muscle triosephosphate isomerase in guanidinium chloride

1. The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. 2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant. 3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits.     

Kinetcs and decay of fumarase activity of immobilized Brevibacterium ammoniagenes cells for continuous production of L-malic acid

The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells.     

Chicken fumarase. II. Kinetic studies.

The catalysis of the hydration of fumarate and deshydration of L - malate by chicken fumarase was measured spectrophotometrically over a range of substrate concentrations from 4 times 10(-3) M to 8 times 10(-5) M for fumarate and from 8 times 10(-2) M to 10(-3) M for L - malate. For the forward and reverse reactions, linear Lineweaver and Burk plots were obtained. The Michaelis constants and the maximum initial velocities for both substrates were determined and the Haldane relation was found to be obeyed. The effect of pH on activity was investigated over a pH range from 5.5 to 9.0 and the data indicate the presence, in the active site, of two ionizable groups, one in the acidic form and one in the basic form. The values of the ionization constants, determined for the enzyme - substrate complexes, agree closely with the ones obtained for the porcine enzyme. The mode of action of twenty-four structural analogs on the initial velocity of the dehydration of L-malate, by chicken fumarase was examined. From these studies, two regions positively charged appear necessary for the effective binding of the carboxylates of the substrates and competitive inhibitors to the active center. Moreover, the data suggest the presence of an additional group, in the catalytic site of chicken fumarase, that stabilizes the carbon-carbon double bond common to fumarate and its structural analogs. Finally, from the comparison of the kinetic properties of the chicken and pig fumarases, it may be concluded that the catalytic mechanism of the homologous enzymes are very similar, if not identical.     

Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin

Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70°C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58°C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70°C. No acetate kinase activity was observed at 70°C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58°C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70°C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70°C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.     

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).     

Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.     

Purification and properties of pig kidney glutamate dehydrogenase.

Glutamate dehydrogenase from pig kidney has been purified to homogeneity by means of affinity chromatography on matrix bound Cibacron Blue F3G-A and gel chromatography on Sepharose 6B. The enzyme exhibits allosteric properties with the substrates alpha-ketoglutarate, ammonium, and NADH, respectively. GTP is a strong inhibitor which strengthened the cooperative interactions between the ammonium binding sites. ADP as an activator relieves the inhibition by GTP. Like glutamate dehydrogenase from bovine liver, glutamate dehydrogenase from pig kidney shows the ability of self-association, too. The sedimentation coefficient increases from 13.5 S at 0.07 mg protein/ml to 19.4 S at 1.32 mg protein/ml. In the sodium dodecylsulphate gel electrophoresis the enzyme migrates as a single band with a molecular-weight at 51000.     

Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.     

Participation of extracellular fumarase in the utilization of malate in cultured carrot cells

Carrot suspension cells were found to be unable to transport malate directly into the cell but utilized it as a single carbon source in a unique manner -they converted malate extracellularly to fumarate and subsequently used it instead. The uptake of fumarate proved to be inducible and sensitive to pH and protonophore. Immuno-blot experiments using an antibody raised against Arabidopsis fumarase showed that fumarase polypeptide appeared in the medium. Fumarase was not detected in medium when fumarate or glucose was used as a carbon source. The activity of fumarase, which catalyzes the reversible hydration reactions, was induced both in the medium (malate into fumarate, releasing protons) and in the cells (fumarate into malate, requiring protons) and resulted in an increase in the pH gradient across the plasma membrane. The reason for the participation of fumarase in the utilization of malate is discussed.     

Isoelectrofocusing of rat muscle adenylosuccinase.

Adenylosuccinase catalyses the conversion of adenylosuccinic acid to AMP and fumarate. We have developed a coupled enzyme staining procedure applicable to nitrocellulose blots after agarose gel isoelectrofocusing of rat muscle adenylosuccinase. The coupling enzymes, fumarase (fumarate to L-malate) and malic enzyme (L-malate to pyruvate and NADPH), are adsorbed to nitrocellulose prior to blotting. The NADPH, mediated by phenazine methosulfate, converts a tetrazolium salt to its blue formazan. This procedure demonstrated that rat muscle adenylosuccinase consists of three isomeric forms present in similar amounts.     

Enzymes of pentose biosynthesis. The quaternary structure and reacting form of transketolase from baker's yeast

Transketolase from baker's yeast is a dimeric enzyme with a molecular weight of 158,000 ± 4000. Sedimentation velocity and sedimentation equilibrium experiments indicate that the enzyme dissociates at low concentrations (less than 0.1 mg/ml) in the absence of the coenzyme, thiamine pyrophosphate. However, no such dissociation was detected in the presence of coenzyme. Reacting enzyme sedimentation velocity measurements showed that the reacting species of the enzyme is a dimer with an s_20,w of 7.7 S.     

Purification and physical properties of homogeneous initiation factor MP from rabbit reticulocytes.

Initiation factor MP was purified 1570-fold with 67% recovery of total activity present in 0.5 M KCl extracts of rabbit reticulocyte ribosomes. Initiation factor MP forms a ternary complex with Met-tRNAf and GTP or a binary complex with Met-tRNAf alone, the details of which are presented in the accompanying paper (Safer, B., Adams, S. L., Anderson. W. F., and Merrick, W. C. (1975) J. Biol. Chem. 250, 9076-9082). Initiation factor MP was homogeneous by the following criteria: (a) electrophoresis as a single band in gels of 5, 6, 7, 8, 9, and 10% acrylamide; (b) equilibration as a single band during isoelectric focusing; (c) sedimentation as a single symmetrical boundary during sedimentation velocity experiments; (d) linear plots of sedimentation equilibrium data; (e) symmetrical absorbance (at 280 nm) and activity profiles during DEAE-cellulose and Sephadex G-200 chromatography, and (f) symmetrical distribution of initiation factor MP during sucrose density gradient band sedimentation. The molecular weight of the initiation factor MP monomer (0.2 mg/ml) by low speed sedimentation equilibrium was 90,800. Calculations based on the Stokes radius and sedimentation velocity show the existence of relatively stable 90,000-dalton monomers or 180,000-dalton dimers at low (0.1 mg/ml) and high (9.75 mg/ml) concentrations of initiation factor MP, respectively. Electrophoresis in sodium dodecyl sulfate gels indicates that initiation factor MP monomer is composed of two noncovalently linked subunits with molecular weights of 52,000 and 34,000. Despite a relatively normal amino acid composition and an isoelectric point of 6.4, initiation factor MP behaves as a basic protein, eluting from phosphocellulose at 650 mM KCl (pH 7.9). Both ternary complex formation and methionyl-puromycin synthesis co-purify, indicating that a single protein is required for both activities.     

X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation

Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G-955-C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.