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PubMed1978

Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from porcine liver. Interaction between the dehydrogenase and cyclohydrolase activities of the multifunctional enzyme.

Methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5) one of the activities of a trifunctional folate-dependent enzyme from porcine liver, uses an ordered kinetic mechannism as determined from initial velocity, product inhibition and dead-end inhibition studies. The final product released from the dehydrogenase is methenyltetrahydrofolate. However, from the time course of appearance of products it is observed that the methenyltetrahydrofolate, rather than equilibrating with the solution, is converted preferentially to formyltetrahydrofolate by the cyclohydrolase, (5,10-methenyletrahydrofolate 5-hydrolase (decyclizing), EC 3.5.4.9) demonstrating a functional interaction between these two enzymic activites.

Aminohydrolases↗

PubMed1976

Renaturation of formyltetrahydrofolate synthetase from urea and guanidinium chloride solutions.

Formyltetrahydrofolate synthetase from Clostridium cylindrosporum and Clostridium acidi-urici was denatured in 6 M urea and 4 M guanidinium chloride. Viscometric, fluorimetric and ultracentrifugal measurements were used to determine that the protein is completely unfolded under these conditions. The polypeptide chains refold upon dilution of the denaturant-protein solutions to give final concentrations of 0.5 M urea or 0.1 M guanidinium chloride. In the presence of NH4+, but not in its absence, the refolded proteins associate to produce the catalytically active tetramer. Refolding and reassociation were followed by measuring changes in protein fluorescence and by determination of sedimentation constants. Under most conditions 80% of the enzymic activity is recovered.

Binding Sites↗

PubMed1970

Inhibition of RNA polymerase and formyltetrahydrofolate synthetase activity by 6-chloro-8-aza-9-cyclopentylpurine. Structure-activity relationships.

The structural requirements for inhibition of bacterial RNA polymerase and rabbit liver formyltetrahydrofolate synthetase activity by a series of purine nucleoside analogs related to 6-chloro-8-aza-9-cyclopentylpurine (689) were investigated. To achieve an inhibitory effect, preincubation of the enzyme preparations with the purine analogs, prior to assay of enzyme activity, was required. The greatest inhibition was produced by analogs containing all three alterations of the purine nucleoside structure: the 6-halo, 8-aza, and 9-cyclopentyl groups. It is suggested that 689 inhibits the activity of enzymes involved in nucleic acid synthesis by a site-directed alkylation.

Journal Article↗

PubMed1982

Assembly of formyltetrahydrofolate synthetase monomers. Kinetic evidence for a temperature-induced conformational change.

The assembly of Clostridium acidi-urici formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) monomers induced by low concentrations of CsCl to form the catalytically active tetramer shows a pronounced lag phase in the time-course progress curve. Preincubation of the monomer for 20 min at 20 degree C in a medium of either high ionic strength, 10% dimethylsulfoxide or 1 M glycerol removes the lag period. Shorter preincubation periods produce the same effect if the medium contains both high ionic strength and dimethylsulfoxide or glycerol. Preincubation at 0 degree C in the presence of these agents has little effect. The reassociation reaction is first-order with respect to monomer concentration, before preincubation at 20 degree C, but second-order after. It is proposed that two conformers of the monomer exist in equilibrium in a temperature-dependent reaction and that the conformer able to reassociate is more favored at 20 degree C than at 0 degree C. High ionic strength or the presence of the organic solvents shift the equilibrium far in the direction of the conformer capable of reassociation, changing the rate-limiting step from a first-order conformational step to a second-order monomer association step.

Clostridium↗

PubMed1976

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae.

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.

Drug Resistance, Microbial↗

PubMed1993

Identification of 10-formyltetrahydrofolate, tetrahydrofolate and 5-methyltetrahydrofolate as major reduced folate derivatives in rat bile.

Reduced folate derivatives in rat bile were examined using high-performance liquid chromatography with electrochemical detection (HPLC-ED). Three peaks of folate compounds were observed on the chromatograms. From the retention-time profiles and hydrodynamic voltammograms, and the profiles of ultraviolet (UV) absorbance spectra obtained by HPLC with photodiode array detection, these 3 peaks were identified as 10-formyltetrahydrofolate (10-HCO-H4PteGlu), tetrahydrofolate (H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H4PteGlu). The rates of bile secretion of 10-HCO-H4PteGlu, H4PteGlu and 5-CH3H4PteGlu were 314 +/- 181, 321 +/- 179 and 449 +/- 198 ng/h (mean +/- S.D.), respectively. 10-HCO-H4PteGlu and H4PteGlu together wtih 5-CH3-H4-PteGlu are found to be the major folate derivatives in rat bile. The nonmethylated folates, 10-HCO-H4PteGlu and H4PteGlu, may also play an important role in folate homeostasis.

Animals↗

PubMed2002

Site-directed mutagenesis of a potential catalytic and formyl phosphate binding site and substrate inhibition of N10-formyltetrahydrofolate synthetase.

Structural studies of N(10)-formyltetrahydrofolate synthetase (FTHFS) have indicated the involvement of Arg 97 in the binding of the formyl phosphate intermediate. Two site-directed mutants were constructed to test this hypothesis: R97S (Ser substitution) and R97E (Glu substitution). The k(cat) of R97S was approximately 60% that of the wild-type enzyme and had K(m) for ATP and formate twofold higher than those of wild type. R97E was completely inactive and had a K(m) for ATP nearly six times that of wild type. Substrate inhibition by tetrahydrofolate was shown to occur in wild-type and R97S enzymes using both steady-state and transient-state kinetic approaches. These results lend greater insight into the mechanistic function of FTHFS by confirming the interaction of both ATP and formate with Arg 97 and introducing the aspect of substrate inhibition by tetrahydrofolate with regard to substrate binding and dissociation.

Adenosine Triphosphate↗

PubMed2003

Transport of 5-formyltetrahydrofolate into primary cultured rat astrocytes.

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured rat astrocytes was studied. Uptake of 5-FTHF into astrocytes was linear in the first 60 min and is saturable with K(m)=3.3 microM and V(max)=27.5 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF displayed the characteristics of countertransport. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methtrexate, and folic acid (K(i)=3.8, 2.7, and 18.4 microM, respectively). Uptake was significantly decreased by sodium azide but was increased by high concentration of sodium cyanide and low concentration of sodium arsenate. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4(')-diisothiocyanostilbene-2,2(')-disulfonic acid. Acute exposure of the cells to ethanol (100mM) inhibited the uptake for 90 min of the experimental duration. It is concluded that astrocytes have a system for the uptake of 5-FTHF and folate analogs which is carrier mediated, this system is sensitive to energy inhibitors and alcohol exposure.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2003

Transport of 5-formyltetrahydrofolate into primary cultured cerebellar granule cells.

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured cerebellar granule cells (CGC) was studied. Uptake of 5-FTHF into CGC was saturable with K(m)=2.86 microM and V(max)=40.8 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF in the astrocytes has a similar style in the time curve. Uptake of 5-FTHF is characterized by countertransport because adding unlabeled 5-FTHF in the medium resulted in the efflux of labeled 5-FTHF. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methotrexate and folic acid (K(i)=6.64, 7.69, and 19.38 microM, respectively). Uptake was significantly decreased by high concentrations of sodium azide and sodium arsenate but not by sodium cyanide. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Acute exposure of the cells to ethanol (100 mM) did not affect the uptake. It is concluded that CGC have a carrier-mediated system for the uptake of 5-FTHF and other folates.

Animals↗

PubMed1997

Folate and 10-formyltetrahydrofolate dehydrogenase (FDH) expression in the central nervous system of the mature rat.

10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH) is a folate-binding protein that is important in folate metabolism. In addition, 10-FTHFDH catalyzes the rate-limiting step in hepatic folate-dependent formate oxidation. We measured folate concentrations and examined cellular 10-FTHFDH expression in regions of the adult rat central nervous system (CNS), to study components of CNS oxidative formate metabolism. Folate was detected in every CNS region studied; the concentrations ranged from 3 to 14% of that detected in the liver. Immunohistochemical expression of 10-FTHFDH was identified in many CNS structures. 10-FTHFDH was mostly expressed by glia, especially astrocytes and ventricular ependyma. Neuronal expression was weak but evident in the cerebral cortex, basal ganglia, cerebellum, and spinal cord. Thus, CNS tissue has the chief components of folate-dependent formate oxidation and the chief site of this oxidation appears to be glia.

Age Factors↗

PubMed2000

Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica.

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures. The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified. Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does. Neither binding site is located in the active site. The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding. Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules. The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions. The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.

Aspartic Acid↗

PubMed1982

Monovalent cation induced reassociation of formyltetrahydrofolate synthetase monitored by Rayleigh light scattering and enzymic activity.

It has been previously established that formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum is reversibly dissociated and inactivated in the absence of certain monovalent cations. In the present paper, the reassociation of monomeric, inactive enzyme to form tetrameric, active enzyme was monitored by Rayleigh light scattering and enzymic activity. Light-scattering measurements confirmed that the active enzyme is composed of four subunits of equal weight. With the assumption that the results of analytical ultracentrifugation are correct--that monomers and tetramers are the only species ever present at appreciable levels--the amount of tetramer formed during reassociation was calculated from the light-scattering data. Evidence for the accumulation of catalytically active intermediates was obtained by comparing the rate of association of monomers (detected by light scattering) to the rate of return of enzymic activity. The accumulation of intermediates was most strikingly seen at low monovalent cation concentration at low ionic strength. Evidence is also presented that sedimentation favors reassociation of the enzyme. The reassociation data were fit to a second-order reversible rate equation. Interestingly, although the data were derived from the same experiments, the kinetic plot based on light-scattering measurements yielded a straight line function with an abrupt change in slope about 10 min after initiation of reassociation, while plots based on enzymic activity measurements gave a single slope.

Cations, Monovalent↗

PubMed1983

Nuclear magnetic resonance studies of formyltetrahydrofolate synthetase interactions with formate and methylammonium ion.

Using nuclear magnetic resonance techniques, we have measured the internuclear distances separating the nucleotide-bound metal from the carbon and hydrogen nuclei of formate as well as the carbon of methylammonium cation when bound to formyltetrahydrofolate synthetase. Measurements were made of the paramagnetic effect on the spin-lattice relaxation rates (1/T1) of 13C and 1H nuclei arising from the replacement of Mg2+ with Mn2+, which binds to the enzyme in the form of a metal-nucleotide complex. Distances from Mn2+ to the formate carbon and proton were found to be 6.3 and 7.4 A, respectively, in the E . ATP . Mn2+ . formate complex and 6.0 and 7.1 A, respectively, in the E . ADP . Mn2+ . formate complex. When tetrahydrofolate was added to the latter complex, the exchange of formate was greatly reduced and became rate limiting for relaxation. These results are consistent with substantial conformational effects produced by the binding of the cofactor. The distance from Mn2+ to the methylammonium carbon in the E . ADP . Mn2+ . CH3NH+3, E . ADP . Mn2+ . formate . CH3NH3+, and E . ADP . Mn2+ . tetrahydrofolate . CH3NH3+ complexes was estimated to be in the range of 7.4-12 A. However, in the E . ADP . Mn2+ formate . tetrahydrofolate . CH3NH3+ complex, the data suggest that exchange of cation contributes significantly to relaxation. These results, combined with other known features of the enzyme, suggest that there may be a monovalent cation site within the active site of the enzyme.

Clostridium↗

PubMed1986

Substrate and inhibitor activities of the screw sense isomers of metal-nucleotide complexes in the formyltetrahydrofolate synthetase reaction.

Phosphorothioate analogues of ATP and isomers of CrATP and CrADP were used to examine the nucleotide stereoselectivity of formyltetrahydrofolate synthetase from procaryotic and eucaryotic sources. Substrate activity of the thio-ATP analogues increased as the site of sulfur substitution was changed from the gamma to the alpha position. Thus, adenine nucleotide analogues substituted with sulfur at an alpha nonbridging position (ATP alpha S isomers) were the most active, and ATP gamma S was inactive. When Mg2+ was used as the divalent cation, both enzymes showed a clear preference (higher V/Km value) for the Sp isomer of ATP beta S although the magnitude of the preference was greater with the bacterial enzyme. With Cd2+ as the divalent cation the Rp isomer was preferred, but the difference was greater with the yeast enzyme. Both (Sp)-MgATP beta S and (Rp)-CdATP beta S have the delta or right-hand screw sense configuration of the metal chelate ring. The reversal of stereoselectivity when the cation was changed indicates that the metal ion is coordinated to the beta-phosphate group. No stereoselectivity was observed when ATP alpha S isomers were used in the presence of Mg2+ or Cd2+, suggesting that the metals are not coordinated to the alpha-phosphate. ATP beta S was also found to be a competitive inhibitor of MgATP and CdATP, and the lowest Ki values were obtained with the lambda screw sense isomers. The screw sense isomers of bidentate CrATP exhibited no detectable substrate activity but were competitive inhibitors of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1990

Primary structure of the thermostable formyltetrahydrofolate synthetase from Clostridium thermoaceticum.

The complete nucleotide sequence of the Clostridium thermoaceticum formyltetrahydrofolate synthetase (FTHFS) was determined and the primary structure of the protein predicted. The gene was 1680 nucleotides long, encoding a protein of 559 amino acid residues with a calculated subunit molecular weight of 59,983. The initiation codon was UUG, with a probable ribosome binding site 11 bases upstream. A putative ATP binding domain was identified. Two Cys residues likely to be involved in subunit aggregation were tentatively identified. No characterization of the tetrahydrofolate (THF) binding domain was possible on the basis of the sequence. A high level of amino acid sequence conservation between the C. thermoaceticum FTHFS and the published sequences of C. acidiurici FTHFS and the FTHFS domains of the Saccharomyces cerevisiae C1-THF synthases was found. Of the 556 residues shared between the two clostridial sequences, 66.4% are identical. If conservative substitutions are allowed, this percentage rises to 75%. Over 47% of the residues shared between the C. thermoaceticum FTHFS and the yeast C1-THF synthases are identical, 57.4% if conservative substitutions are allowed. Hydrophobicity profiles of the C. acidiurici and C. thermoaceticum enzymes were very similar and did not support the idea that large hydrophobic domains play an important role in thermostabilizing the C. thermoaceticum FTHFS.

Amino Acid Sequence↗

PubMed1979

Folylpoly-gamma-glutamates as cosubstrates of 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase.

N10-Formyltetrahydropteroylpoly-gamma-glutamates (N10-formyl-H4PteGlun) having n = 1, 3, 4, 5, 6, and 7 glutamyl residues have been tested as cosubstrates of the purine biosynthesis enzyme 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase (AICAR transformylase) of chicken liver. The cosubstrates were synthesized by solid-phase synthesis, reduced catalytically, and formylated; a purified enzyme preparation was used and assayed spectrophotometrically following the deltaOD at 298 nm resulting from conversion of the formylated folate to the free tetrahydro form. Km values and Vm values determined at saturating concentrations of AICAR and at 25 and 150 mM KC1 were used to calculate the relative specificity constants Vm/Km for the N10-formyl-H4PteGlun. At physiologic [K+] (150 mM) they were 1.0, 52, 250, 93, 120, and 59 and at the lower (25 mM) [K+] the relative specificity constants were 1.0, 64, 78,34, 48, and 37 for n = 1, 3, 4, 5, 6, and 7, respectively. The poly-gamma-glutamates are clearly the preferred cosubstrates, particularly when tested at physiologic [K+]. The maximal relative specificity constant observed with N10-formyl-H4PteGlu4 supports the hypothesis that regulation of certain pathways of one-carbon metabolism may operate via alterations of the poly-gamma-glutamyl chain length. No inhibition by the unnatural (d) isomers of the N10-formyl-H4PteGlun was observed.

Acyltransferases↗

PubMed1975

The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits: a solvent isotope effect.

In the presence of specific monovalent cations (K+, Cs+, NH4+), inactive monomers of formyltetrahydrofolate synthetase associate to a catalytically active tetramer. The rate and extent of association of enzyme monomers prepared from C. cylindrosporum are enhanced 3.3-and about 50-fold, respectively, by the substitution of D2O for H2O. Both rate and equilibrium solvent isotope effects are due to a decrease in D2O of the dissociation constant of the monomer-cation complex. Analysis of rate and equilibria data obtained in solvent mixtures of varying deuterium/protium ratios indicates that the isotope effect may be due to the change in bonding of a single monomer proton during the association process. The data are most consistent with a model in which this proton is in a very weak potential in the cation-free monomer and is converted to a "normal" water-like proton in the monomer-cation complex.

Ammonia↗

PubMed2000

The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica.

The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10)-formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface.

Amino Acid Sequence↗