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PubMed1975

Electron paramagnetic resonance and water proton relaxation rate studies of formyltetrahydrofolate synthetase-manganous ion complexes. Evidence for involvement of substrates in the promotion of a catalytically competent active site.

Conformational properties of the active site of formyltetrahydrofolate synthetase from Clostridium cylindrosorum have been examined by EPR spectroscopy and by solvent proton relaxation rate (PPR) studies of manganous complexes with the enzyme. Ternary enzyme-Mn-nucleotide complexes give EPR spectra which are very similar to those for the binary Mn-nucleotide complexes. However, upon addition of tetrahydrofolate to form the quaternary complexes, enzyme-MnADP-tetrahydrofolate and enzyme MnATP-tetrahydrofolate the EPR line shapes are changed substantially. Spectra for the quaternary complexes exhibit narrow line widths, and the splitting patterns are characteristic of a slightly asymmetric electronic environment for the bound Mn(II). Addition of formate to the ADP quatenary complex induces a further significant narrowing of the EPR line widths, although in the absence of tetrahydrofolate, formate does not influence the EPR spectrum for the enzyme-MnADP species. Both Pi and nitrate cause changes in the EPR patterns for the higher complexes of the enzyme which involve both ADP and tetrahydololate. However, the Pi effect is not influenced by the presence of formate whereas the characteristic effect of nitrate is potentiated only when formate is present. EPR sectra for the thernary complex with the beta, gamma-methylene analog of ATP App(CH2)p differ significantly from spectra for the binary App(CH)p complex is not influenced by further additions of tetrahydrofolate and of tetrahydorfolate and formate. The failure of spectra for the App(CH)p complex to respond to additions of the other substrates for the reaction is in marked contrast to the behavior found for the natural nucleotide substrates and is tentatively attributed to the lack of a protein-mediated interaction between the nucleotide and tetrahydrofolate binding sites in the analog complex. The frequency dependence of solvent PRR in the presence of the various complexes allows an estimate of the correlation times for electron-nuclear dipolar interaction and thereby the extent of hydration of the bound Mn(II) among the various complexes..

Adenosine Diphosphate↗
PubMed1991

Effects of diastereoisomers of 5-formyltetrahydrofolate on cellular growth, sensitivity to 5-fluoro-2'-deoxyuridine, and methylenetetrahydrofolate polyglutamate levels in HCT-8 cells.

We investigated the biological activities of the natural and unnatural diastereoisomers of 5-formyltetrahydrofolate [(6S)- and (6R)-5-HCO-H4PteGlu, respectively, both 99.99% pure], using a human ileocecal carcinoma cell line (HCT-8). Optimal cell growth could be supported by (6S)-5-HCO-H4PteGlu at concentrations as low as 1 nM. (6R)-5-HCO-H4PteGlu did not support growth. Modulation of the in vitro cytotoxicity of 5-fluoro-2'-deoxyuridine (FdUrd) and intracellular (6R)-5,10-methylenetetrahydrofolates [(6R)-CH2H4PteGlun] pools by (6S)- and (6R)-5-HCO-H4PteGlu was determined with cells growing in 1 nM (6S)-5-HCO-H4PteGlu. For the control cells, the concentration of FdUrd inhibiting growth by 50% was 179 nM and the total (6R)-CH2H4PteGlun was 2.3 pmol/10(6) cells. When cells were treated with (6S)-5-HCO-H4PteGlu for 24 h, the 50% inhibition concentration of FdUrd decreased with increasing concentrations of (6S)-5-HCO-H4PteGlu, and reached a plateau of 36 nM when (6S)-5-HCO-H4PteGlu was greater than or equal to 1 microM. The total (6R)-CH2H4PteGlun pools were augmented by (6S)-5-HCO-H4PteGlu dose dependently up to 6.8 pmol/10(6) cells at 1 microM (6S)-5-HCO-H4PteGlu. (6S)-5-HCO-H4PteGlu at 10 microM did not further increase the total (6R)-CH2H4PteGlun, but induced a marked shift in the polyglutamate chain length distribution, with an increase in tri- and tetra-, and a decrease in penta-, hexa-, and heptaglutamate. The down-shift of (6R)-CH2H4-PteGlun polyglutamate chain length observed after (6S)-5-HCO-H4PteGlu treatment did not impair the modulation of FdUrd cytotoxicity. Thus shorter chain (6R)-CH2H4PteGlua (n = 3-4) function as well as longer ones (n = 5-7). (6R)-5-HCO-H4PteGlu, at 200 microM, had no effect on the cytotoxicity of FdUrd, the total (6R)-CH2H4PteGlun level, or chain length distribution in the presence or absence of additional (6S)-5-HCO-H4PteGlu. These results suggest that the high plasma (6R)-5-HCO-H4PteGlu concentrations (up to 200 microM) achieved in patients following i.v. administration of high doses of (6R,S)-5-HCO-H4PteGlu probably do not have adverse effects on the modulation of antitumor activity of FdUrd or 5-fluorouracil. Since the optimal dose and schedule of (6S)-5-HCO-H4PteGlu for modulation of fluoropyrimidines may vary from one cell type to another, introducing high doses of (6R,S)-5-HCO-H4PteGlu in patients so that the plasma concentration of the natural isomer reaches 10 microM is still recommended.

Cell Division↗
PubMed1978

5-Methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid (folinic acid), and folic acid requirements of normal and Rous sarcoma virus-infected chicken fibroblasts.

Normal and Rous sarcoma virus-infected chicken fibroblasts proliferate maximally in a culture medium containing a physiological (10 ng/ml) concentration of 5-methyltetrahydrofolic acid or folinic acid (5-formyltetrahydrofolic acid), while their maximal proliferation requires a hyperphysiological (1000 ng/ml) concentration of folic acid. The normal and Rous-infected fibroblasts do not differ in their requirements for 5-methyltetrahydrofolate, folinic acid, or folic acid.

Animals↗
PubMed1989

Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-formyltetrahydrofolate polyglutamates.

Methenyltetrahydrofolate synthetase (EC 6.3.3.2) catalyzes the irreversible ATP and Mg2+-dependent transformation of 5-formyltetrahydrofolate (N5-HCO-H4-pteroylglutamic acid (PteGlu] to 5,10-methenyltetrahydrofolate. The physiological function of this reaction remains unknown even though it is potentially involved in the intracellular metabolism of the large doses of N5-HCO-H4-PteGlu (leucovorin) administered to cancer patients. We have tried to elucidate methenyltetrahydrofolate synthetase's physiological role by examining the consequences of its inhibition in MCF-7 human breast cancer cells by the folate analog 5-formyltetrahydrohomofolate (fTHHF), a potent competitive inhibitor with a Ki of 1.4 microM. fTHHF inhibited MCF-7 cell growth with an IC50 of 2.0 microM during 72-h exposures, and this effect was fully reversible by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. A correlation was observed between increases in intracellular N5-HCO-H4-PteGlu concentrations following fTHHF and cell growth inhibition. De novo purine synthesis was inhibited at the second folate-dependent enzyme, phosphoribosyl aminoimidazole-carboxamide formyltransferase (AICAR transferase; EC 2.1.2.3), as determined by aminoimidazole carboxamide rescue and azaserine inhibition studies. N5-HCO-H4-PteGlu pentaglutamate was a potent inhibitor of purified MCF-7 cell AICAR transferase with a Ki of 3.0 microM while the monoglutamate was not an inhibitor up to 10 microM and fTHHF was only weakly inhibitory with a Ki of 16 microM. These findings suggest that methenyltetrahydrofolate synthetase activity is needed to prevent de novo purine synthesis inhibition by N5-HCO-H4-PteGlu polyglutamates.

Acyltransferases↗
PubMed1989

Chromosomal localization of the gene for the human trifunctional enzyme, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.

A trifunctional protein in man, 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of tetrahydrofolate derivatives; these derivatives supply one-carbon units for intermediary metabolism. Somatic cell hybridization and in situ hybridization were used to localize the functional gene coding for this protein--to human chromosome 14q24, near the c-fos and TGF-beta 3 loci. A second hybridizing sequence, possibly a pseudogene, was identified near the centromere of the X chromosome, at Xp11.

Aminohydrolases↗
PubMed1988

Primary structure of a human trifunctional enzyme. Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.

A DNA clone complementary to the messenger RNA encoding the human trifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyl-tetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase has been isolated from a lambda gt10 library. In vitro transcription-translation of the 3.1-kilobase cDNA clone yields a protein of 101 kDa, which is of identical size and exhibits the same immunoreactivity as the enzyme purified from human liver. A coding region of 2805 base pairs in the cDNA encodes a protein of 935 amino acids. The initiator methionine is absent from the purified enzyme and the amino-terminal 30 amino acids derived by automated sequence analysis are identical (arginine at position 18 was not identified) with that deduced from the nucleotide sequence. The amino acid sequence of the human enzyme shows extensive homology with that of the yeast enzyme, although the amino-terminal bifunctional dehydrogenase-cyclohydrolase domain is less homologous than the carboxyl-terminal synthetase domain. A region was identified which probably serves as a link between these two major domains of the human enzyme. The synthetase domain contains two regions that are homologous to consensus sequences for an ATP-binding site.

Amino Acid Sequence↗
PubMed1987

Pharmacokinetics of leucovorin (D,L-5-formyltetrahydrofolate) after intravenous injection and constant intravenous infusion.

The pharmacokinetics of the active and inactive diastereoisomers of leucovorin and its active metabolite, 5-methyltetrahydrofolate (5-CH3-THF) were studied after iv injection of leucovorin in normal human subjects at a dose of 28 mg/m2, and in patients given 500 mg/m2 daily by constant iv infusion for a 5.5 day period. In both studies the plasma half-life (t1/2) of the active isomer, L-formyltetrahydrofolate (CHO-THF), was only 32 to 35 minutes, whereas the inactive isomer, D-CHO-THF had a plasma t 1/2 of 352 to 485 minutes. During constant infusion, the plasma levels reached plateaus of 2.33 and 37.5 microM for L-CHO-THF and D-CHO-THF, respectively. The inactive isomer was cleared from plasma only by urinary excretion of the unchanged drug. The active isomer was also excreted unchanged in the urine but in addition was extensively metabolized to the active metabolite L-5-CH3-THF. The active metabolite achieved a plasma level of 4.85 microM during constant infusion and appeared to have a longer t 1/2 after constant infusion than was observed after iv injection. Furthermore a larger apparent volume of distribution (Vd) of 5-CH3-THF was obtained in the constant infusion study. These findings suggest that constant iv infusion of large doses of leucovorin can considerably expand the intracellular pools of active folate. The consequence of the extensive accumulation of the inactive isomer, D-CHO-THF, is not known. However, the small Vd of D-CHO-THF suggests that it does not extensively accumulate in tissues.

Biotransformation↗
PubMed1985

In vitro synthesis of the trifunctional protein, methylenetetrahydrofolate dehydrogenase--methenyltetrahydrofolate cyclohydrolase--formyltetrahydrofolate synthetase, in normal and transformed cells.

The trifunctional eucaryotic protein, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of folate derivatives which are required for thymidine and purine synthesis. The protein was synthesized in vitro using a reticulocyte lysate cell-free translation system. The immunoprecipitated translation product programmed by normal rat liver and rat brain mRNA was identical in size to that programmed by mRNA from transformed cells (HeLa, Morris hepatamas 5123D and 3924A). The protein synthesized by normal rat liver slices or Reuber H35 hepatoma cells in culture was similar in size to the translation product, suggesting that there is no significant posttranslational modification in vivo that alters the molecular weight. Differences in the level of synthesis were observed among the tissues examined. Since no consistent pattern emerged, however, it appears that transformation per se does not alter the structure or expression of this polypeptide. Nonetheless, a two- to three-fold increase in synthesis by Morris hepatoma 5123D may be an important biological observation with respect to studying the regulation of this protein that is critical for nucleotide synthesis.

Aminohydrolases↗
PubMed1982

Methotrexate rescue by 5-methyltetrahydrofolate or 5-formyltetrahydrofolate in lymphoblast cell lines.

The rescue of lymphocytes from methotrexate (MTX) growth inhibition by 5-methyltetrahydrofolate (5-methyl-THF) and 5-formyltetrahydrofolate (5-formyl-THF) has been studied. Rescue by 5-methyl-THF is selective for cells with high levels of homocysteine:5-methyl-THF methyl-transferase (methyltransferase). At MTX concentrations which inhibited growth greater than or equal to 85% in both leukemic T-lymphocytes (CCRF-CEM) and Epstein-Barr-transformed B-lymphocytes (LAZ-007), 5 micro M 5-formyl-THF rescued more effectively than did 5-methyl-THF, in either the presence or absence of the methyltransferase inhibitor, nitrous oxide. At less inhibitory MTX concentrations, both reduced folates rescued equally, except when methyltransferase was inhibited by nitrous oxide in which case 5-formyl-THF was clearly superior. In the absence of nitrous oxide, both cell lines contained approximately equal amounts of methyltransferase. Some apparent differences in the rescue of these cell lines with 5-methyl-THF were attributable to their different sensitivity to MTX. When metabolism of reduced folates was severely impaired by MTX and nitrous oxide, lymphocytes were rescued with 5-[methyl-14C]methyl-THF, and the uptake of 14C into DNA was measured. In corporation was very low, indicating that cellular oxidation of 5-methyl-THF to 5,10-methylene-tetrahydrofolate is minimal even under forcing conditions. MTX selectively in vivo will be influenced by the level of methyltransferase in tumor and normal tissues.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1982

Formyltetrahydrofolate synthase from Clostridium thermoaceticum. An electron microscopic study and specific interaction of the enzyme with ATP and ADP.

Formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium thermoaceticum is a 240 000-molecular-weight, tetrameric enzyme composed of identical subunits. When negatively stained with uranyl acetate or sodium phosphotungstate the enzyme appears as two distinct projections when viewed in the electron microscope. One of these, resembling the letter "H", exhibits four flexible arms, while the other, called a "twin", has a compact projection. The twin projection appears to represent the native enzyme in solution. It consists of two outstanding intensity maxima separated by a narrow bridge of dense stain, with each of these intensity maxima being divided into the two submaxima by a second bridge of stain running perpendicular to the first one. The outstanding intensity maxima represent dimers, whereas the submaxima represent a single subunit. The volume calculated from the electron microscopy projections for the dimer is 157 nm3, which corresponds to a Mr of 127 000 or 254 000 for the tetrameric enzyme. The H projections reflect enzyme particles artificially flattened and "opened" during the drying of the negatively stained enzyme. The four arms of the H each represent a subunit. The appearance of H projections is prevented by either using a deep-stain procedure, which offers a mechanical preservation of the native enzyme structure, or by the binding of ATP, ADP or adenosine 5'-[beta, gamma-methylene]triphosphate. These nucleotides, when binding to the enzyme, cause a conformational change. This was observed by ultraviolet and visible circular dichroism and absorption spectrometry with the native enzyme and an enzyme labeled with fluorescein mercuric acetate attached to a sulfhydryl group of each subunit.

Adenosine Diphosphate↗
PubMed1980

Chemical cross-linking stabilizes the enzymic activity and quaternary structure of formyltetrahydrofolate synthetase.

Clostridium cylindrosporum formyltetrahydrofolate synthetase tetramers cross-linked with dimethyl suberimidate remained active in the absence of the monovalent cations normally required for enzymic activity and the tetrameric conformation. The modified enzyme was analyzed by sodium dodecyl sulfate electrophoresis, sedimentation velocity, and gel permeation chromatography. Under the experimental conditions used, the enzyme was only partially cross-linked; 74% of the enzyme was cross-linked dimer or monomer. Nonetheless, the modified enzyme is able to retain enzymic activity and the tetrameric structure under conditions where native enzyme would be completely dissociated and inactivated. The result suggests that cross-linked dimers strongly associate with each other and with monomers. Flame emission spectroscopy indicates that cross-linked enzyme contains two monovalent cations per tetramer.

Ammonia↗
PubMed1977

Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. A multifunctional protein from porcine liver.

Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase from porcine liver have been co-purified more than 500-fold to apparent homogeneity. The inability of three sequential chromatographic procedures followed by affinity chromatography using NADP+- or ATP-substituted Sepharose to resolve the three activities demonstrates that they are physically associated. Molecular weight estimates of the native protein by gel filtration (Mr = 150,000) and by dodecyl sulfate gel electrophoresis (Mr = 100,000) indicate that the native structure is probably a single subunit. Since only one protein band is seen on dodecyl sulfate gels, it is concluded that the three activities are properties of a single polypeptide chain. The kinetic properties of the three activities are described, the most unusual feature being the susceptibility of the cyclohydrolase to competitive inhibition by NADP+, NAD+, ATP, and folate.

Aminohydrolases↗
PubMed1976

Formyltetrahydrofolate synthetase-catalyzed formation of ATP from carbamyl phosphate and ADP. Evidence for a formyl phosphate intermediate in the enzyme's catalytic mechanism.

Formyltetrahydrofolate synthetase from Clostridium cylindrosporum catalyzes phosphoryl transfer from carbamyl phosphate in ADP to form ATP. The phosphoryl transfer reaction has an obligatory requirement for tetrahydrofolate presumably as a cofactor for a proper conformation of the active site. Carbamyl phosphate is an analog of formyl phosphate-a potential intermediate in the normal enzymic reaction. The ability of the enzyme to promote synthesis of ATP from carbamyl phosphate and ADP supports a stepwise chemical reaction mechanism for the enzyme in which formyl phosphate participates as a tightly bound intermediate.

Adenosine Diphosphate↗
PubMed1996

Folate and 10-formyltetrahydrofolate dehydrogenase in human and rat retina: relation to methanol toxicity.

Ocular toxicity, a well-recognized outcome of methanol poisoning in humans, correlates with formate accumulation in blood following the ingestion of methanol. Rodents, however, are a species known to be resistant to the toxicity of formate. The present study was designed to determine whether components of folate-dependent formate oxidation, e.g., folate and 10-CHO-H4-folate dehydrogenase (10-FDH), exist in retina and whether differences in these components might explain species-determined susceptibility to methanol intoxication. Total folate levels were determined in human and rat retinal tissues and were found to be much lower than the levels in liver. However, folate levels in human retina were only 14% of those determined in rat retina. Western blot analysis was used to quantify the amount of cytosolic and mitochondrial 10-FDH in retinal tissues. Results of these studies showed that comparable amounts of this enzyme were present in both cellular compartments in each species. However, the amount of 10-FDH in human retina was approximately three times the amount found in rat retina. In order to determine the cell type(s) within the retina that contained 10-FDH, immunohistochemical staining for 10-FDH was carried out. Confocal microscopic image analysis of human and rat retinae showed colocalization of 10-FDH primarily with Muller cell protein markers [glial fibrillary acidic protein (GFAP), vimentin, and carbonic anhydrase]. Therefore, 10-FDH was found to be preferentially localized in this cell type. Since Müller cells appear to represent the target for formate-induced ocular toxicity, our data suggest that formate oxidation reactions might serve two roles, first a protective role and then a role in methanol-induced toxicity in Müller cells.

Adenosine Triphosphate↗
PubMed1994

Detection and quantification of 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) in rat retina, optic nerve, and brain.

Methanol poisoning is characterized by the accumulation of formic acid, a metabolite of methanol, which can lead to metabolic acidosis and ocular toxicity. Formate metabolism to CO2 is governed by tissue H4folate and 10-FTHFDH levels. Presumably, rats are not normally susceptible to formate toxicity because they possess high hepatic H4folate and 10-FTHFDH levels. However, the ability of target tissues to metabolize formate is not known. Therefore, studies were performed to determine whether 10-FTHFDH was present in rat retina, optic nerve, and brain. 10-FTHFDH levels were determined using Western blot analysis of mitochondrial and postmitochondrial preparations from these tissues. Hepatic mitochondrial and postmitochondrial levels of 10-FTHFDH were 13 and 12 ng/micrograms protein, respectively. Postmitochondrial levels of 10-FTHFDH in rat retina, optic nerve and whole brain were 0.2, 1.3, and 2.1 ng/micrograms protein; mitochondrial values in retina and brain were 0.2 and 1.5 ng/micrograms protein, respectively. Postmitochondrial values obtained for rat brain regions were similar to those found for whole brain. These results suggest that, in rats, target tissues possess the capacity to metabolize formate to CO2 and may be protected from formate toxicity through this folate-dependent system.

Animals↗