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We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.
The mechanism of the reaction catalyzed by N10-formyltetrahydrofolate synthetase involves the formation of formyl phosphate as an intermediate which then formylates tetrahydrofolate at the N-10 position. Previous studies demonstrated that the non-enzymic formylation of tetrahydrofolate by formyl phosphate occurs exclusively at the more nucleophilic 5-nitrogen in the reduced pyrazine ring. The experiments described in this report were designed to determine whether N5-formyltetrahydrofolate might be the first product to be formed on the enzyme, followed by formyl transfer to the 10-nitrogen via the cyclic intermediate N5,10-methenyltetrahydrofolate. If this were the case, oxygen from solvent H2O would be incorporated into the formyl group of the N10-derivative. By conducting the reaction in a 1:1 mixture of [16O]H2O and [18O]H2O and using 13C NMR spectroscopy we show that no 18O is incorporated into the product and conclude that the reaction proceeds via a direct formylation of the N-10 position by formyl phosphate.
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10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Previous studies of 10-formyltetrahydrofolate dehydrogenase purified from rat or pig liver homogenized in phosphate buffers indicated the presence of copurifying 10-formyltetrahydrofolate hydrolase activity, which catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate and formate. We find that the supernatant from rat liver homogenized in mannitol/sucrose/EDTA medium contains essentially all of the total cellular 10-formyltetrahydrofolate dehydrogenase activity, but no measurable hydrolase activity. Treating mannitol/sucrose/EDTA-washed mitochondria with Triton X-100 (0.5%) releases hydrolase activity in soluble form. 10-Formyltetrahydrofolate dehydrogenase purified from the mannitol/sucrose/EDTA supernatant has no 10-formyltetrahydrofolate hydrolase activity. Results of kinetic experiments using the hydrolase-free dehydrogenase give a complex rate equation with respect to (6R,S)-10-formyltetrahydrofolate. Double-reciprocal plots fit a 2/1 hyperbolic function with apparent Km values of 3.9 and 68 microM. Our results indicate that 10-formyltetrahydrofolate hydrolase and dehydrogenase are not alternate catalytic activities of a single protein, but represent two closely related and separately compartmentalized hepatic enzymes.
The racemic mixture, [6RS]-5-formyltetrahydrofolate, is widely used clinically. In human subjects, orally-administered pure unnatural C-6 isomers, [6R]-5-formyltetrahydrofolate and [6S]-5,10-methenyltetrahydrofolate, were recently shown to be metabolized to the natural isomer, [6S]-5-methyltetrahydrofolate. We re-analysed the data from human studies published during the past four decades in which oral doses (< or =10 mg) of racemic mixtures of these folates were used. We re-evaluated the data to determine whether these racemic mixtures are only 50 % bioactive or, as we now predict, more than 50 % bioactive. Our analyses indicate that, in human subjects, oral doses of the racemic mixture of the two formyltetrahydrofolates are 20-84 % more bioactive than would be predicted. These data are consistent with the following pathway: chemical conversion of these folates to 10-formyltetrahydrofolate; oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate; subsequent enzymic conversion of 10-formyldihydrofolate to dihydrofolate by 5-amino-4-imidazolecarboxamide ribotide transformylase; and finally the well-established metabolism of dihydrofolate to [6S]-5-methyltetrahydrofolate. An additional review of the literature supports the in vivo oxidation of 10-formyltetrahydrofolate occurring to a certain extent, as 10-formyl-folic acid is rapidly formed after the administration of folic acid (pteroylglutamic acid) or 5-formyltetrahydrofolate in human subjects. The dogma that an oral dose of the unnatural C-6 isomer of 5-formyltetrahydrofolate is not bioactive in human subjects does not withstand scrutiny, most probably due to the previously unrecognized in vivo oxidation of 10-formyltetrahydrofolate. This discovery unveils new folate metabolism in human subjects.
The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.
1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7--0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.
A highly sensitive, radioenzymatic method has been developed for the specific and quantitative estimation of (S)-5-formyltetrahydrofolate. This method is based on enzymatic cycling of the 5-formyl derivative to methylenetetrahydrofolate followed by entrapment into a stable ternary complex with thymidylate synthase and tritiated fluorodeoxyuridylate. Determination of bound radiolabeled ligand permits estimation of the original folate. The initial cycling step is catalyzed by the enzyme, methenyltetrahydrofolate synthetase, which is specific for the (S)-diastereomer of 5-formyltetrahydrofolate and generates a product which can be further cycled to tetrahydrofolate using either 10-formyltetrahydrofolate deacylase or glycinamide ribonucleotide transformylase. Tetrahydrofolate is ultimately converted to the entrapable methylene derivative in the presence of excess formaldehyde. Using this assay recovery of reference (S)-5-formyltetrahydrofolate was linear over the range 0.03-1.9 pmol with an average recovery of 83 +/- 2%. The method has been applied to estimation of plasma (S)-5-formyltetrahydrofolate from a volunteer who had been administered (R,S)-5-formyltetrahydrofolate. Where comparison was possible, estimation of plasma (S)-5-formyltetrahydrofolate by this one step ternary complex-based method yielded results that were very similar to those observed by Straw et al. (Cancer Res., 44, 3114, 1984) who used an HPLC-based method for separation of diastereomeric mixtures of reduced folates and microbiological growth dependence to determine (S)-5-formyltetrahydrofolate.
The ability of human blood in vitro, and partially purified red blood cells, to metabolize leucovorin, or 5-formyltetrahydrofolate, has been examined. A radioenzymatic assay based upon entrapment of 5,10-methylenetetrahydrofolate, and other reduced folates after cycling to this form, into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxyuridine-5'-monophosphate was used to estimate reduced folate metabolites. Incubation of whole blood samples with (R,S)5-formyltetrahydrofolate resulted in a time- and concentration-dependent extracellular accumulation of the reduced folates, 5-methyltetrahydrofolate, tetrahydrofolate, 10-formyltetrahydrofolate, and 5,10-methylenetetrahydrofolate. While accumulation with time was nonlinear, the tetrahydrofolate pool showed the greatest overall increase in concentration. 5-Methyltetrahydrofolate, which was the only reduced folate detected in plasma prior to introduction of (R,S)5-formyltetrahydrofolate, accumulated more slowly than tetrahydrofolate. 10-Formyltetrahydrofolate and 5,10-methylenetetrahydrofolate accumulated even more slowly but exhibited nonlinear kinetic patterns similar to those of tetrahydrofolate and 5-methyltetrahydrofolate. When blood cells were removed by centrifugation, a complete loss of metabolic activity was observed. Exposure of purified red blood cells to (R,S)5-formyltetrahydrofolate resulted in accumulation of extracellular reduced folates that was similar to that in whole blood samples while partially purified white blood cells exhibited little activity. Metabolism of the (S) diastereomer of 5-formyltetrahydrofolate accounted for essentially all of the observed extracellular accumulation of reduced folates. We propose that red blood cell-mediated metabolism of 5-formyltetrahydrofolate could, in part at least, account for reduced folate accumulation in plasma when leucovorin is administered to humans.
It has been assumed that humans cannot utilize 5,6,7,8-tetrahydrofolates with the unnatural configuration at carbon 6, since these folates are enzymatically and microbiologically inactive. We hypothesized that orally administered unnatural [6R]-5-formyltetrahydrofolate or [6S]-5,10-methenyltetrahydrofolate is bioactive in humans. Subjects were given independent oral doses of these unnatural folates and of a natural [6S]-5-formyltetrahydrofolate. Plasma, before and after the dose for 4 h, and 2 h urine were collected. Areas under the curve for the change in plasma folate concentrations were measured microbiologically and urinary folates were measured using HPLC. Based on findings of plasma and urinary folates, the unnatural folates were estimated to be 14-50% active as compared to [6S]-5-formyltetrahydrofolate. The major plasma and urinary folate was [6S]-5-methyltetrahydrofolate in all experiments. In urine, a [6S]-5-formyltetrahydrofolate peak was observed only after a [6S]-5-HCO-H4folate dose and peaks of unnatural [6S]-10-formyltetrahydrofolate and 5-formyltetrahydrofolate were identified after a [6R]-5-formyltetrahydrofolate dose. A possible pathway that explains our findings is discussed. This pathway includes the oxidation of the unnatural [6S]-10-formyltetrahydrofolate to 10-formyl-7,8-dihydrofolate which can be further metabolized by 5-amino-4-imidazolecarboxamide-ribotide transformylase producing dihydrofolate. Dihydrofolate can then be metabolized to [6S]-5-methyltetrahydrofolate by well-established metabolism.
We have characterized the uptake of 5-formyltetrahydrofolate into isolated rat liver mitochondria. Uptake of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and folic acid was linear for the first 2 min of incubation. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate were accumulated in mitochondria to 3.7- and 4.3-fold, respectively. 5-Formyltetrahydrofolate transport showed a pH optimum at 5.5 and was saturable with an apparent Km = 2.80 mumol.L-1 and Vmax = 3.49 pmol.min-1.mg protein-1. Uptake of 5-formyltetrahydrofolate was inhibited by the structural analog 5-methyltetrahydrofolate and, to a much lesser extent, folic acid and methotrexate. Uptake was not inhibited by various substrates of known mitochondrial transporters, e.g., succinate, malate, citrate, glutamate and phosphate, nor was uptake inhibited by the sulfhydryl reagent, N-ethylmaleimide. Uptake was not stimulated by preloading of mitochondria with malate or aspartate. Uptake of 5-formyltetrahydrofolate was not affected by the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone nor by collapsing the electrical gradient with valinomycin. These results indicate that 5-formyltetrahydrofolate uptake into mitochondria via a carrier-mediated system specific for the reduced, naturally occurring folates. The folates, once in the mitochondria, may be converted to the polyglutamylated forms, which will not readily diffuse out of the mitochondria.
This report describes studies designed to evaluate possible inhibitory effects of diaminoantifolates on folate-dependent biosynthetic enzymes in intact L1210 leukemia cells. A novel approach is described which involves an assessment of the metabolism of and biosynthetic flux of the one-carbon moiety from (6S)5-formyltetrahydrofolate in folate-depleted cells. Pretreatment with methotrexate (10 microM), resulting in the formation of methotrexate polyglutamates, or continuous incubation with trimetrexate (1 microM) inhibited growth of folate-depleted L1210 cells in the presence of folic acid or 5-formyltetrahydrolate. In both control and drug-treated cells, double-labeled (6S)-5-[14C]formyl[3H]tetrahydrofolate was rapidly metabolized with the loss of the [14C]formyl group. Under all conditions, the predominant metabolite was 10-formyl[3H]tetrahydrofolate, detectable both intracellularly and extracellularly. In drug-treated cells, there was a remarkably small decrease in the level of 10-formyl[3H]tetrahydrofolate (approximately 30%) and a 10-fold rise in the level of [3H]dihydrofolate to less than 20% of the total folate pool. The incorporation of [14C]formyl group from 5-[14C]formyltetrahydrofolate into thymidylate, serine, and methionine was unaffected by the presence of 1 microM trimetrexate, consistent with the generation of sufficient 5,10-[14C]methylenetetrahydrofolate to drive these reactions. Similarly, the presence of methotrexate polyglutamates had no effect at the level of amino acid synthesis; however, carbon transfer into thymidylate was markedly inhibited. Even though 10-formyltetrahydrofolate was readily formed from 5-formyltetrahydrofolate in this model, the net incorporation of 14C from 5-[14C]formyltetrahydrofolate into purine nucleotides was inhibited by both methotrexate and trimetrexate treatments. Similar findings were obtained when [14C]glycine incorporation into purine nucleotides was monitored in cells incubated with unlabeled 5-formyltetrahydrofolate. Finally, in antifolate-treated cells incubated with unlabeled 5-formyl-tetrahydrofolate, transfer of 14C from [14C]formate or [14C]serine into biosynthetic products or incorporation of [3H]deoxyuridine into nucleic acids was potently inhibited. These results suggest that insufficient levels of tetrahydrofolate and 5, 10-methylenetetrahydrofolate were formed to drive these reactions despite the presence of high levels of 10-formyltetrahydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)
A new rapid procedure for purifying 10-formyltetrahydrofolate dehydrogenase results in 90 mg of pure enzyme from two rabbit livers. This abundant liver enzyme is known to bind its product tetrahydropteroylpentaglutamate (H4PteGlu5) so tightly that it does not dissociate during size exclusion chromatography. 10-Formyltetrahydrofolate dehydrogenase is also known to exhibit strong product inhibition by H4PteGlu5. There is a several-fold excess of 10-formyltetrahydrofolate dehydrogenase subunits in liver relative to the concentration of H4PteGlun, suggesting that in vivo this enzyme may bind significant amounts of this coenzyme in a nearly irreversible enzyme. H4PteGlu5 complex. How this tightly bound H4PteGlun is transferred to the other two enzymes in the cytosol, serine hydroxymethyltransferase and C1-tetrahydrofolate synthase, which use H4PteGlu5 as a substrate, is the subject of this investigation. Analysis of the product inhibition curve for 10-formyltetrahydrofolate dehydrogenase shows that H4-PteGlu5 has a dissociation constant near 15 nM which is 60-fold lower than the Ks for 10-formyl-H4PteGlu5. Fluorescence titration studies also yield a Kd of about 20 nM for H4PteGlu5. Coupling the 10-formyltetrahydrofolate dehydrogenase reaction to an excess of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase not only abolishes product inhibition but also increases the initial rate of its activity by about 2-fold. Passage of a reaction mixture of 10-formyltetrahydrofolate dehydrogenase down a size exclusion column results in enzyme with 1 equiv of H4PteGlu5 bound per subunit. However, addition of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase results in a rapid transfer of this bound folate to these enzymes. Evidence is also presented that the tightly bound folate is in equilibrium with solvent H4PteGlu5.
Single (13)C6-labelled doses of pteroylmonoglutamic acid (PteGlu; 634 nmol) or 5-formyltetrahydrofolic acid (431-569 nmol) were given to fasted adult volunteers, and the rise in total and (13)C-labelled plasma 5-methyltetrahydrofolic acid metabolite monitored over 8 h by HPLC and liquid chromatography-MS. The dose-adjusted area under the curve (AUC) for total (labelled plus unlabelled) plasma 5-methyltetrahydrofolic acid following a 5-formyltetrahydrofolic acid test dose was 155 % that obtained following a PteGlu test dose. Surprisingly, an average 60 and 40 % of the total plasma 5-methyltetrahydrofolic acid response to [(13)C6]PteGlu and [(13)C6]5-formyltetrahydrofolic acid, respectively, was unlabelled; an observation never before reported. Short-term kinetics of plasma [(13)C6]5-methyltetrahydrofolic acid showed a slower initial rate of increase in plasma concentration and longer time to peak following an oral dose of [(13)C6]PteGlu compared with that for an oral dose of [(13)C6]5-formyltetrahydrofolic acid, while the [(13)C6]5-methyltetrahydrofolic acid AUC for [(13)C6]5-formyltetrahydrofolic acid was 221 % that for [(13)C6]PteGlu. These data indicate that PteGlu and 5-formyltetrahydrofolic acid, which are thought to be well absorbed (about 90 %) at physiological doses, exhibit dramatically different rates and patterns of plasma response. A limitation in the rate of reduction of PteGlu before methylation could result in slower mucosal transfer of [(13)C6]5-methyltetrahydrofolic acid derived from [(13)C6]PteGlu into the plasma. This, when coupled with an observed similar plasma clearance rate for [(13)C6]5-methyltetrahydrofolic acid metabolite derived from either folate test dose, would yield a comparatively smaller AUC. These findings suggest potential problems in interpretation of absorption studies using unlabelled or labelled folates where the rate of increase, the maximum increase, or the AUC, of plasma folate is employed for test foods (mainly reduced folates) v. a 'reference dose' of PteGlu.
The metabolic role of 5-formyltetrahydrofolate is not known; however, it is an inhibitor of several folate-dependent enzymes including serine hydroxymethyltransferase. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme known to metabolize 5-formyltetrahydrofolate and catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. In order to address the function of 5-formyltetrahydrofolate in mammalian cells, intracellular 5-formyltetrahydrofolate levels were depleted in human 5Y neuroblastoma by overexpressing the human cDNA encoding MTHFS (5YMTHFS cells). When cultured with 2 mM exogenous glycine, the intracellular serine and glycine concentrations in 5YMTHFS cells are elevated approximately 3-fold relative to 5Y cells; 5YMTHFS cells do not contain measurable levels of free methionine and display a 30-40% decrease in cell proliferation rates compared with 5Y cells. Medium supplemented with pharmacological levels of exogenous folinate or methionine ameliorated the glycine induced growth inhibition. Analysis of the folate derivatives demonstrated that 5-methyltetrahydrofolate accounts for 30% of total cellular folate in 5Y cells when cultured with 5 mM exogenous glycine. 5YMTHFS cells do not contain detectable levels of 5-methyltetrahydrofolate under the same culture conditions. These results suggest that 5-formyltetrahydrofolate inhibits serine hydroxymethyltransferase activity in vivo and that serine synthesis and homocysteine remethylation compete for one-carbon units in the cytoplasm.
One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.
5-Formyltetrahydrofolate was found to reverse the binding of methotrexate to dihydrofolate reductase in the Ehrlich ascites tumor in vitro. When cells pretreated with methotrexate were resuspended in methotrexate-free buffer containing 5-formyltetrahydrofolate (or 5-methyltetrahydrofolate), net dissociation of the antifolate from the enzyme was observed. Methotrexate associated with the enzyme under these conditions was below the enzyme binding capacity. However, glucose or azide increased the fraction of dihydrofolate reductase associated with methotrexate and abolished the effect of tetrahydrofolates on this intracellular component. Addition of 5-fluoro-2'-deoxyuridine had no effect on this response to the reduced folate, thereby precluding a direct role for the thymidylate synthase-dependent generation of dihydrofolate in this dissociation of methotrexate from dihydrofolate reductase. Enzyme-bound methotrexate could also be reduced by exposure to 5-formyltetrahydrofolate prior to uptake and efflux of free methotrexate. When cells were incubated under conditions which favored formation of methotrexate polyglutamate derivatives, subsequent treatment with 5-formyltetrahydrofolate had no effect on the binding of the conjugated antifolate to dihydrofolate reductase. These findings support a role for dihydrofolate reductase as a locus for competitive binding interactions between reduced folates and methotrexate that may be a basis for the ability of 5-formyltetrahydrofolate to prevent the biochemical effects of this antifolate. These data suggest that the presence of methotrexate polyglutamate derivatives and cellular energy metabolism may be critical determinants of the responsiveness of methotrexate-treated cells to reduced folates and may play important roles in the selectivity of 5-formyltetrahydrofolate rescue.