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PubMed2004

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.

Aldehydes↗

PubMed1996

Quantification of the carbon flow through the folate-dependent one-carbon pool using radiolabeled histidine: effect of altered thyroid and folate status.

Hyperthyroidism and folate deficiency are known to alter the concentration of hepatic folate pools and the activity of a number of enzymes related to one-carbon metabolism. To evaluate the physiological significance of this relationship on a functional basis, we have developed and utilized an in vivo tracer kinetic technique to quantify the carbon flow through the one-carbon pool as a function of thyroid and folate status. Control, hyperthyroid, and folate-restricted rats were continuously infused with L-[ring-2-14C]histidine to measure the oxidative flow of carbon from histidine through the one-carbon pool to CO2. As expected, the hepatic activities of a number of enzymes involved in the catabolism of histidine to CO2 were markedly decreased in hyperthyroid (histidase, 69%; urocanase, 30%; 10-formyltetrahydrofolate dehydrogenase, 65%) and folate-restricted (10-formyltetrahydrofolate dehydrogenase, 44%) rats. In addition, folate-restricted animals exhibited a 63% decrease in the hepatic concentration of total reduced folates. However, tracer kinetic analysis indicated an enhanced catabolism of histidine: the carbon flux from histidine to CO2 was increased approximately threefold in hyperthyroid rats and twofold in folate-restricted animals. Thus, in the case of hyperthyroidism and dietary folate restriction, changes in static measurements such as metabolite concentrations and enzyme activities do not reflect the dynamic tracer kinetic assessment of the carbon flux that is actually occurring in vivo. The kinetic data also demonstrate that the percentage of total entry into the 10-formyltetrahydrofolate pool originating from histidine catabolism was almost threefold greater compared to 5-methyltetrahydrofolate pool.

Animals↗

PubMed1980

The role of folates in methanol toxicity.

In the monkey and human, methanol toxicity is characterized by a metabolic acidosis and an ocular toxicity which occur coincident with an accumulation of formate in blood. In contrast, methanol insensitive species such as the rat do not accumulate formate after methanol administration. Folate-dependent reactions are involved in the oxidation of formate to CO2 in both the rat and the monkey. Monkey liver contains a significantly lower hepatic folate level than does rat liver and, thus, formate accumulation in the monkey may be related to a functional folate deficiency in this species. Formate metabolism in the monkey can be stimulated with folate administration. After methanol administration, treatment of monkeys with repetitive doses of either 5-formyltetrahydrofolic acid or folic acid results in a marked decrease in blood formate accumulation, an absence of metabolic acidosis and no blood bicarbonate depletion. Also, methanol toxicity, once established in the monkey, can be reversed with 5-formyltetrahydrofolic acid administration. The results indicate that 5-formyltetrahydrofolic acid decreases formate accumulation after methanol by stimulating the rate of formate oxidation or utilization and provide additional evidence for the involvement of folate-dependent reactions in the metabolism and toxicity of methanol in the monkey.

Animals↗

PubMed1988

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate.

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗

PubMed1989

Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver.

The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase (EC 2.1.1.13), on the subcellular distribution of hepatic folate coenzymes was determined. In controls, cytosolic folates were 5-methyltetrahydrofolate (45%), 5- and 10-formyltetrahydrofolate (9 and 19%, respectively), and tetrahydrofolate (27%). Exposure of rats to an atmosphere containing 80% nitrous oxide for 18 h resulted in a marked shift in this distribution pattern to 5-methyltetrahydrofolate, 84%; 5- and 10-formyltetrahydrofolate, 2.1 and 9.1%, respectively; and tetrahydrofolate, 4.7%. Activity of the cytosolic enzyme, methionine synthetase, was reduced by about 84% as compared to that of air breathing controls. In controls, mitochondrial folates were 5-methyltetrahydrofolate (7.3%), 5- and 10-formyltetrahydrofolate (11.5 and 33.1%, respectively), and tetrahydrofolate (48.1%). This distribution did not change after exposure to nitrous oxide. These results show that the effects of nitrous oxide inactivation of vitamin B12 are confined to the cytosol, at least in the short term, and suggest that there is little, if any, transport of free folates between the cytosolic and mitochondrial compartments.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1990

Growth inhibition by homofolate in tumor cells utilizing a high-affinity folate binding protein as a means for folate internalization.

A subline (JT-1) of L1210 mouse leukemia cells that contains elevated levels of a high-affinity folate binding protein is sensitive to growth inhibition by homofolate. Inhibition was observed at nanomolar concentrations of folate or 5-formyltetrahydrofolate where the high-affinity binding protein is the predominant uptake route for folate compounds. At 1.0 nM folate, inhibition of growth by 50% occurred at 0.7 nM homofolate, and maximal inhibition exceeded 90% at homofolate concentrations above 10 nM. Homofolate also inhibited the uptake of 1.0 nM [3H]folate by L1210/JT-1 cells in 72-hr cultures, and the extent of uptake inhibition by 1.0 and 20 nM homofolate was comparable to the inhibition of cell growth by the same concentrations of homofolate. At a growth-limiting concentration of 5-formyltetrahydrofolate (0.5 nM), half-maximal inhibition of L1210/JT-1 cell growth occurred at 1.0 nM homofolate. When excess concentrations of folate (5 microM) or 5-formyltetrahydrofolate (0.5 microM) were added to the medium, no growth inhibition was observed for homofolate at concentrations up to 100 microM. Parental cells lacking the folate binding protein did not respond to homofolate either at growth-limiting (0.5 microM) or excess (5.0 microM) levels of folate. Binding measurements showed that homofolate has a high affinity for the folate-binding protein (Ki = 0.03 nM) but interacts poorly with the reduced-folate transport system (Ki = 203 microM). These results indicate that homofolate inhibits the growth of L1210 cells when intracellular folates are acquired via the high-affinity folate binding protein. The basis for this inhibition appears to be competition by homofolate for substrate binding and internalization.

Animals↗

PubMed1991

Compartmentation of intracellular folates. Failure to interconvert tetrahydrofolate cofactors to dihydrofolate in mitochondria of L1210 leukemia cells treated with trimetrexate.

Following exposure of L1210 leukemia cells to antifolates, tetrahydrofolate-dependent purine and pyrimidine biosyntheses are blocked despite the presence of the major portion of tetrahydrofolate cofactors. Previous studies from this laboratory demonstrated that this cannot be due to direct inhibition of thymidylate synthase by dihydrofolate polyglutamates or other endogenous folates and suggested that this phenomenon is due to compartmentation of tetrahydrofolate cofactors unavailable for interconversion and/or oxidation when dihydrofolate reductase activity is abolished by antifolates. The present paper evaluates the possibility that tetrahydrofolate cofactors in subcellular organelles, in particular, mitochondria, are unavailable for oxidation by thymidylate synthase. Particulate and cytosolic fractions were obtained from L1210 cells following homogenization and differential centrifugation. The crude mitochondrial fraction contained 20.1% of the total folate pool and included 5-formyltetrahydrofolate, 10-formyltetrahydrofolate and tetrahydrofolate in proportions similar to intact cells. The cytosolic fraction had an increased proportion of tetrahydrofolate and decreased proportions of 5-formyl- and 10-formyltetrahydrofolate relative to intact cells or the particulate fraction. Exposure of cells to 10 microM trimetrexate for 30 min produced approximately 45% interconversion of tetrahydrofolate cofactors to dihydrofolate in the cytosolic fraction, a level much greater than that observed in whole cell extracts (25-30%), but had no effect on folate pools in the crude mitochondrial fraction. These data indicate that subcellular compartmentation accounts, in part, for the failure to oxidize tetrahydrofolate cofactors to dihydrofolate in the presence of antifolate levels that abolish dihydrofolate reductase activity.

Animals↗

PubMed1995

Identification and characterization of human mitochondrial methenyltetrahydrofolate synthetase activity.

We present evidence for the presence of the folate metabolism enzyme methenyltetrahydrofolate synthetase (MTHFS) in mitochondria. MTHFS activity was identified in the matrix of mitochondria purified from human liver biopsies. Mitochondrial and cytoplasmic MTHFS specific activities are similar, 85% of the total cellular MTHFS activity is in the cytoplasm and both native enzymes have similar molecular weights (approximately 25 kDa). Studies using purified mitochondrial MTHFS from CA46 human Burkitt lymphoma cells reveal that mitochondrial MTHFS behaves kinetically like the cytoplasmic enzyme with Km values of 4.7, 0.8 and 22 microM respectively for (6R,S)-5-formyltetrahydrofolate monoglutamate, (6S)-5-formyltetrahydrofolate pentaglutamate and ATP. This finding adds to previous observations that various folate-dependent enzymes reside in the mitochondria of eucaryotic cells. Intracellular tetrahydrofolate metabolism is highly compartmentalized and mitochondrial MTHFS activity is necessary for the entry of mitochondrial 5-formyltetrahydrofolate into the mitochondrial folate pool.

Burkitt Lymphoma↗

PubMed1970

Effects of thyroxine on the synthesis of folate coenzymes in rat liver.

1. The effects of thyroidectomy and of ;acute' and ;chronic' administration of thyroxine on the synthesis of folate coenzymes were studied by determining the liver contents of folate active derivatives and the enzymic activities involved in their biosynthesis. The effect of thyroxine on the same enzymes in vitro was also studied. 2. In thyroidectomized rats the liver contents of folate coenzymes did not change except for a slight decrease in the contents of 5-formyltetrahydrofolate and tetrahydrofolate compared with those in control rats. 3. In the same animals serine hydroxymethyltransferase and formyltetrahydrofolate synthetase activities decreased markedly. 4. The ;chronic' administration of thyroxine to thyroidectomized rats caused more evident variations in the liver contents of folate coenzymes and in particular a decrease in the contents of 5-formyltetrahydrofolate, tetrahydrofolate, 5(or 10)-formyl derivatives of tetrahydropteroylpolyglutamate and of 5(or 10)-formyl derivatives of pteroylpolyglutamate. 5. The enzymic activities did not show significant variations. 6. The ;acute' administration of thyroxine caused changes in the liver contents of some folate derivatives such as 10-formyldihydrofolate, 10-formylfolate, tetrahydrofolate and the 10-formyl derivative of dihydropteroylpolyglutamate. In these animals also the enzymic activities were unchanged. 7. No effect of thyroxine on enzymic activities in vitro was observed.

Animals↗

PubMed1996

Methotrexate alters carbon flow through the hepatic folate-dependent one-carbon pool in rats.

The chemotherapeutic value of methotrexate resides in its ability to perturb folate-dependent one-carbon metabolism and subsequently inhibit DNA synthesis. To assess the functional effect of methotrexate on hepatic one-carbon metabolism, we have developed and applied tracer kinetic techniques in vivo to quantify the carbon flux through the folate-dependent one-carbon pool in rats. Following a 7-day treatment period with methotrexate (0.2 mg/kg body weight), the tracers L-[ring-2-14C] histidine and L-[methyl-3H] methionine were simultaneously infused into control and methotrexate-treated rats. Methotrexate treatment decreased hepatic tetrahydrofolate, methyltetrahydrofolate, and formyltetrahydrofolate, concentrations by 63, 83, and 58%, respectively. Furthermore, the enzymatic activity of 10-formyltetrahydrofolate dehydrogenase, the terminal enzyme in the catabolism of the ring-2-carbon of histidine to CO2, was diminished by 32% in methotrexate-treated animals. These changes in enzyme activity and folate coenzyme concentrations did not result in a significant decrease in the oxidative flow of carbon from histidine to CO2 in methotrexate-treated rats compared to control animals (2.40 and 3.22 micromol/h/kg3/4, respectively). Oxidative carbon flow was reflective of tetrahydrofolate and formyltetrahydrofolate pools when expressed as a percent of total folate: neither coenzyme pool was diminished as a result of methotrexate treatment. In contrast, the reductive carbon flux through the one-carbon pool from histidine to methionine was significantly decreased 59% in methotrexate-treated (7.63 micromol/h/kg3/4) versus control rats (18.73 micromol/h/kg3/4)). Likewise, methyltetrahydrofolate, as a percent of total folate, was reduced 51% in methotrexate-treated rats. Consequently, total measured carbon flow (oxidative+reductive) was 54% lower in rats subjected to subchronic methotrexate treatment. These tracer kinetic experiments quantitatively demonstrate the extent to which methotrexate alters the actual carbon flow through the hepatic folate-dependent one-carbon pool, primarily directed at diminishing the reductive carbon flow towards methyltetrahydrofolate and methionine synthesis.

Animals↗

PubMed1991

Biological activity of racemic folate mixtures fed to folate-depleted rats.

Most commercially available tretrahydro and substituted folates are racemic mixtures of R and S isomers about pteridine-carbon 6. Some reports show the unnatural stereoisomers modify specific aspects of folate metabolism, whereas others show no significantly different biologic effects between isomers. The extent to which unnatural folate stereoisomers are inert in vivo is unclear. Possible interactions between isomers at normal dietary folate intakes were tested by comparing growth, liver and serum folate concentrations, and congener patterns of folate-depleted rats fed amino acid-based diets with 283, 566 or 1132 nmol folic acid (standards), or 566 nmol racemic 5-methyltetrahydrofolate, 5-formyltetrahydrofolate or 5,10-diformyltetrahydrofolate/kg diet for 3 wk. Growth and tissue folate levels increased with each increment in dietary folic acid. Growth and liver and serum folate concentrations of rats fed 566 nmol 5-formyltetrahydrofolate or 5-methyltetrahydrofolate/kg diet were similar to those of rats fed 283 nmol folic acid/kg diet. Rats fed 5,10-diformyltetrahydrofolate lost weight and had depressed serum folate levels, and most died. Results show that biologic activity of racemic 5-formyltetrahydrofolate and 5-methyltetrahydrofolate was half that of folic acid, and the R isomer did not affect growth at the levels fed. Addition of a second formyl group destroyed folate bioactivity. The folate-depleted rat model is useful for testing biologic effects of individual folate stereoisomers in vivo as they become commercially available.

Administration, Oral↗

PubMed2005

Differential kinetic behavior and distribution for pteroylglutamic acid and reduced folates: a revised hypothesis of the primary site of PteGlu metabolism in humans.

Single (13)C(6)-labeled doses of pteroylmonoglutamic acid (PteGlu: 634 nmol; n = 14), (6S-)5-formyltetrahydrofolic acid (431-569 nmol; n = 16), or [(15)N(1-7)]-intrinsically labeled spinach (mainly 5-methyltetrahydrofolate) (588 nmol; n = 14) were fed to fasting adult volunteers. Plasma-labeled 5-methyltetrahydrofolic acid responses were monitored for 8 h. There was a slower rate of increase in plasma-labeled 5-methyltetrahydrofolic acid and longer time to peak (171 +/- 9 min; mean +/- SEM) following an oral dose of [(13)C(6)]PteGlu than either [(13)C(6)]5-formyltetrahydrofolic acid (54 +/- 10 min) or [(15)N(1-7)]spinach folate (60 +/- 13 min) suggesting saturated metabolic capacity for the biotransformation of PteGlu. Mathematical modeling generated a significantly higher mean "apparent absorption" for 5-formyltetrahydrofolic acid (38%) and spinach folate (44%) than for PteGlu (24%). The high "relative absorption" of reduced folates to PteGlu was unexpected given that PteGlu itself, from (14)C-tracer mass balance experiments, is almost completely absorbed. Although it is ubiquitously accepted that a physiological dose of PteGlu is reduced and methylated in the epithelial cells of the small intestine, and that essentially only 5-methyltetrahydrofolic acid is exported into the hepatic portal vein (HPV), as is the case for absorbed reduced 1-carbon-substituted folates, modeling indicated greater liver sequestration when PteGlu was used as the test dose, suggesting that PteGlu enters the HPV unaltered and that the liver is the primary site of initial metabolism. Because of the observed differential plasma response and the hypothesized difference in the site of initial metabolism, the historical use of PteGlu as a "reference folate" in studies of folate bioavailability is seriously questioned.

Erythrocytes↗

PubMed1978

The mechanism of 5-methyltetrahydrofolate transport by human erythrocytes.

The mechanism involved in 5-methyltetrahydrofolate uptake by human cells is poorly understood. To more clearly elucidate this physiologically important process, transport of the vitamin was studied in human erythrocytes. 5-methyltetrahydrofolate uptake was found to increase with reticulocytosis, but measurable incorporation occurred in erythrocyte suspensions depleted of reticulocytes, leukocytes, and platelets, indicating uptake by mature erythrocytes. Incubation of erythrocytes with increasing concentrations of [(14)C]5-methyltetrahydrofolate resulted in increasing uptake but decreasing percentage incorporation, consistent with saturation of a carrier system. Both influx and efflux phases of uptake were temperature dependent, with almost no transport at 4 degrees C. Uptake of [(14)C]5-methytetrahydrofolate was effectively inhibited by unlabeled 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but not by pteroylglutamic acid. Prior incubation with 5-formyltetrahydrofolate increased uptake of [(14)C]5-methyltetrahydrofolate, and extracellular 5-formyltetrahydrofolate enhanced efflux of [(14)C]5-methyltetrahydrofolate. Nearly total depletion of ATP increased uptake of [(14)C]5-methyltetrahydrofolate, but efflux was unchanged. Column chromatography of membrane-free hemolysate after incubation with [(14)C]5-methyltetrahydrofolate showed 95% of radioactivity corresponded to marker radioisotope, and no other peak was noted. Thus peripheral erythrocytes incorporate 5-methyltetrahydrofolate by a saturable, temperature-dependent, substrate-specific process which is influenced by counter-transport. This mechanism is qualitatively similar to the carrier-mediated transport of folate compounds previously described in other cell types. Therefore, human erythrocytes should be useful for detailed characterization of this membrane carrier system.

Anemia, Aplastic↗

PubMed2005

Loss of reduced folate carrier function and folate depletion result in enhanced pemetrexed inhibition of purine synthesis.

Pemetrexed is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown with 25 nmol/L racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 micromol/L pemetrexed, but protection of a reduced folate carrier (RFC)-null subline required both thymidine and hypoxanthine above a concentration of 30 nmol/L pemetrexed. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased, and was further diminished when HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC(50), in a HeLa-derived line 8-fold resistant to pemetrexed due to a modest increase in TS. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, pemetrexed inhibition of GARFT increases. These data support the concept that the contribution to pemetrexed activity by inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines.

Animals↗

PubMed1985

Characterization of mammalian phosphoribosylglycineamide formyltransferase from transformed cells.

Phosphoribosylglycineamide formyltransferase levels were studied in several mammalian tissues and were found to be elevated 3- to 6-fold in some anaplastic cells, including rat hepatoma H-35 and mouse leukemia L1210, as compared to their corresponding normal tissues. The enzyme was found to reside in the cytosol of chicken liver and L1210 cells. The enzyme was purified to near homogeneity, as judged by a single band on polyacrylamide gels after electrophoresis in the presence of sodium dodecyl sulfate, from two mammalian sources, mouse L1210 and mouse Sarcoma 180. Comparison of the digestion patterns of L1210 enzyme and chicken liver enzyme upon exposure to chymotrypsin showed some similarity between the two, as did cross-reactivity in Western blots of the chicken enzyme with antibodies raised to the L1210 enzyme. Subunit molecular weight of the L1210 and Sarcoma 180 phosphoribosylglycineamide formyltransferases is about 117,000. Steady-state kinetics was performed with the purified murine enzyme in the presence of 5'-phosphoribosyl-N-glycineamide and 10-formyltetrahydrofolate and with 5'-phosphoribosyl-N-glycineamide and 10-formyl-5,8-deazafolate, establishing that these mammalian enzymes utilize 10-formyltetrahydrofolate as the actual cofactor. 11-Formyltetrahydrohomofolate was found to be an inhibitor of the murine and human (HeLa) enzymes, competitive with respect to 10-formyltetrahydrofolate, with KiS of 1 and 3 microM, respectively. 11-Formyldihydrohomofolate was an inhibitor of HeLa cell growth with a 50% effective dose of 8-11 microM.

Acyltransferases↗

PubMed1984

Purification and properties of 5,10-methenyltetrahydrofolate synthetase from Lactobacillus casei.

5,10-Methenyltetrahydrofolate synthetase (EC 6.3.3.2), which catalyzes the ATP- and Mg2+ -dependent isomerization of 5-formyl- to 5,10-methenyltetrahydrofolate, has been purified 10,000-fold from Lactobacillus casei using sequential affinity chromatography on immobilized 5-formyltetrahydrofolate and ATP. The enzyme is homogeneous when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is monomeric with a molecular mass of 23,000 Da, and contains a high proportion of hydrophobic amino acids and a single cysteine residue. At 30 degrees C, the turnover number is 88 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively. The enzyme is specific for (6S)-5-formyltetrahydrofolate, but ATP can be replaced by other nucleoside 5'-triphosphates with varying efficiency. The purified enzyme is markedly stabilized by the non-ionic detergent, Tween 20.

Amino Acids↗

PubMed1984

5,10-Methenyltetrahydrofolate synthetase. Purification and properties of the enzyme from rabbit liver.

The enzyme 5,10-methenyltetrahydrofolate synthetase has been purified to homogeneity from rabbit liver. The substrates are MgATP and 5-formyltetrahydrofolate. The enzyme will accept as substrates a variety of divalent metal ions and trinucleotides. The products of the reaction are 5,10-methenyltetrahydrofolate, ADP, and inorganic phosphate. The enzyme is a monomeric protein with a molecular weight of about 28,000. The stability of the enzyme is unusually sensitive to pH and ionic strength. Initial velocity studies suggest that the enzyme catalyzes a sequential mechanism. The turnover number of the enzyme is 300/min at 30 degrees C. The Km values for MgATP and 5-formyltetrahydrofolate are 0.3 mM and 0.5 microM, respectively. Product and dead-end inhibition studies indicate that substrates bind to the enzyme by a random mechanism. Vmax and the Km for MgATP are essentially constant over the pH range of 5.0-7.5. The results suggest that the enzyme catalyzes a reaction which serves as a salvage pathway to generate metabolically active one-carbon units from 5-formyltetrahydrofolate.

Adenosine Triphosphate↗

PubMed1984

Role of the cellular oxidation-reduction state in methotrexate binding to dihydrofolate reductase and dissociation induced by reduced folates.

5-Formyltetrahydrofolate promotes the net dissociation of methotrexate bound to dihydrofolate reductase in the Ehrlich ascites tumor (L. H. Matherly et al., Cancer Res., 43: 2694-2699, 1983). Treatment of Ehrlich tumor cells with glucose or inhibitors of electron transfer stabilized the association of the antifolate with dihydrofolate reductase as reflected by a 2-fold increased fraction of dihydrofolate reductase-bound methotrexate and an abolition of the 5-formyltetrahydrofolate-induced dissociation of the inhibitor-enzyme complex. Glucose and azide were also found to increase the intracellular ratio of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP+) in the tumor approximately 8- and 11-fold, respectively. However, other agents which enhanced the association between methotrexate and its target enzyme were less effective in increasing the intracellular level of NADPH relative to NADP+. Micromolar concentrations of NADPH promoted methotrexate binding to the purified Ehrlich tumor dihydrofolate reductase. Bound methotrexate could be dissociated from the purified enzyme by 5-methyltetrahydrofolate but less readily by 5-formyltetrahydrofolate and only in the presence of reduced levels of NADPH relative to NADP+. The tetraglutamate derivative of 5-methyltetrahydrofolate was even more effective than the underivatized compound in dissociating methotrexate from dihydrofolate reductase. These findings suggest a critical role for the cellular oxidation-reduction state in determining the affinity of dihydrofolate reductase for methotrexate and thus the cellular sensitivity to the antifolate. In addition, the data are consistent with the possibility that dihydrofolate reductase is a key locus for intracellular competitive interactions between reduced folates and methotrexate during leucovorin rescue from the pharmacological effects of the antifolate.

Animals↗