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PubMed1982

Folate pentaglutamate and folate hexaglutamate mediated one-carbon metabolism.

The proposal that folate polyglutamate cofactors of different chain lengths function differently in metabolism was investigated. We identified the one-carbon units present in rat liver folates within each of the liver folate polyglutamate groups, the folate penta-, hexa-, and heptaglutamates. This identification revealed that at the pentaglutamate level, 5-methyl-H4folate was the major form, accounting for 18% of the total liver folates, with small amounts of H4folate. At the hexaglutamate level, 5-methyl-H4folate and H4folate were major forms, accounting for 17% and 22% of the liver folates, respectively. At the heptaglutamate level, 5-methyl-H4folate occurred in small amounts while H4folate predominated. The rats used here had been fed a diet low in methionine and were exposed to N2O gas. These results are qualitatively similar to those from rat brain [Brody, T., Shin, Y. S., & Stokstad, E. L. R. (1976) J. Neurochem. 27, 409--413] where 5-methyl-H4folate was a major fraction of the folate pentaglutamates but a minor, if detectable, part of the hexa- and heptaglutamates. The folates 5-methyl-H4PteGlu5-7 were metabolically active in the liver, as illustrated by the severe contraction in the amounts of these folates following an injection of methionine. This indicates that "folate-binding proteins" do not prevent 5-methyl-H4folates from entering into one-carbon metabolism.

Animals↗

PubMed1983

Interaction of tetrahydropteroylpolyglutamates with two folate-dependent multifunctional enzymes.

The naturally occurring pteroylpolyglutamate derivatives are substrates for the folate-mediated reactions in cells, including the reactions catalyzed by two multifunctional folate dependent enzymes in eucaryotes. The appropriate derivatives of tetrahydropteroyl (glutamate)n where n = 1, 3, 5, or 7 were used to determine the specificity for, and kinetic advantages of the extra glutamyl residues with two multifunctional proteins from pig liver: methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, and formiminotransferase-formininotetrahydrofolate cyclodeaminase. Specificity for the polyglutamate derivatives ranged from 10- to 70-fold as indicated from Km values or from the ability to inhibit the five different enzyme activities. With the sequential activities of the transferase-deaminase enzyme, it was demonstrated that when the tetrahydropteroyl pentaglutamate is used as a substrate, the intermediate formimino-compound does not accumulate in the medium. That this kinetic observation is due to preferential transfer of the pentaglutamate- but not monoglutamate intermediate from transferase to deaminase sites without its release from the enzyme molecule was supported by three types of experiments. Chemical modification to yield monofunctional derivatives of the transferase-deaminase affected the kinetics of the recombined activities only with the pentaglutamate substrate, causing a lag in the appearance of final product. Inhibition studies demonstrated that the deaminase activity could preferentially be inhibited only with the monoglutamate substrate. The deaminase activity with the monoglutamate substrate was increased by providing elevated formiminotetrahydrofolate in the assay mixture; no effect was observed when the reaction was carried out with pentaglutamate. Preliminary binding studies indicate a single folate site per subunit of the octameric enzyme, suggesting a type of combined transferase-deaminase site.

Aminohydrolases↗

PubMed2000

Glutamyl hydrolase. pharmacological role and enzymatic characterization.

gamma-Glutamyl hydrolase (GH, EC 3.4.19.9) is a lysosomal and secreted glycoprotein that hydrolyzes the gamma-glutamyl tail of antifolate and folate polyglutamates. Tumor cells that have high levels of GH are inherently resistant to classical antifolates, and further resistance can be acquired by elevations in GH following exposure to this class of antitumor agents. The highest level of expression in normal tissues occurs in the liver and kidney in humans. When panels of tumors are compared with normal tissues, GH expression is elevated in cancerous hepatic and breast tissue. A second poly-gamma-glutamate hydrolyzing enzyme, glutamate carboxypeptidase II, is a transmembrane protein whose active site is on the outside of the cell, occurring in the prostate gland, small intestine, brain, kidney, and tumor neovasculature. It is a high-affinity (nanomolar), low-turnover, zinc co-catalytic enzyme. In contrast, GH is a low-affinity (micromolar), high-turnover enzyme that has a cysteine at the active site. Data are presented suggesting that Cys110 is the nucleophile that attacks the gamma-amide linkage and causes hydrolysis. GH is being evaluated as an intracellular target for inhibition in order to enhance the therapeutic activity of antifolates and fluorouracil.

Animals↗

PubMed1999

Role of folylpolyglutamate synthetase and folylpolyglutamate hydrolase in methotrexate accumulation and polyglutamylation in childhood leukemia.

Inefficient polyglutamylation is a mechanism of resistance to methotrexate (MTX) in childhood T-lineage acute lymphoblastic leukemia (T-ALL) and in acute myeloid leukemia (AML) in comparison with childhood c/preB-ALL. We analyzed the profile of MTX polyglutamylation in childhood c/preB-ALL, T-ALL, and AML (n = 45, 15, and 14, respectively), the activity of the MTX-polyglutamate synthesizing enzyme folylpolyglutamate synthetase (FPGS) (n = 39, 11, and 19, respectively) and of the MTX-polyglutamate breakdown enzyme folylpolyglutamate hydrolase (FPGH) (n = 98, 25, and 34, respectively). MTX-Glu4-6 accumulation after 24 hours exposure to 1 micromol/L [3H]-MTX in vitro was lower in T-ALL (threefold) and AML (fourfold) compared with c/preB-ALL (P </=.001). The FPGS activity was twofold lower in T-ALL and AML than in c/preB-ALL samples (P <. 01). FPGH activity was not different between c/preB-ALL and T-ALL, but threefold higher in AML (P <.001). FPGS, FPGH, and the ratio FPGS/FPGH were correlated with MTX-Glu4-6 accumulation (r =.49, r = -.34 and r =.61, respectively). Multivariate analysis showed that FPGS, but not FPGH, was an independent contributor for MTX-Glu1-6 accumulation, but not for MTX-Glu4-6 accumulation. In conclusion, low FPGS activity is associated with low accumulation of MTX-Glu4-6 in T-ALL and AML. For the group of AML as compared with the group of ALL, a high FPGH activity can play an additional role.

Antimetabolites, Antineoplastic↗

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↗

PubMed1996

Critical factors for optimizing the 5-fluorouracil-folinic acid association in cancer chemotherapy.

BACKGROUND: The 5-fluorouracil (FU)-folinic acid (FA) association has demonstrated clinical efficacy in colorectal cancer, both in adjuvant and metastatic situations. However, there is no clear consensus about the optimal FU-FA schedule and dose. In addition, it would be of interest to identify FU-FA-responsive tumors. DESIGN: Our purpose was to review preclinical and clinical data dealing with prediction of FU-FA sensitivity and optimization of FU-FA schedules. RESULTS: Preclinical studies have highlighted the importance of thymidylate synthase (TS), the cellular target of the FU-FA mechanism of action, for predicting FU sensitivity. It appears that the more sensitive cell lines express the lowest TS activity. Interestingly, the cell lines sensitive to FA supplementation are those more sensitive to FU. The role of TS in FU-FA responsiveness has been clearly demonstrated in patients with colorectal and gastric cancers. Preliminary in vitro and clinical data have shown that the folylpolyglutamate synthetase (FPGS), the enzyme responsible for folate polyglutamylation, is another promising tool for identifying FU-FA-responsive tumors. So far, results of clinical trials do not form a clear consensus regarding the need to administer high FA doses for improving FU-FA treatment. Experimental studies on human cancer cell lines have demonstrated the wide variability among cell lines, ranging from 0.05 to 200 microns, of 1 FA concentrations required for maximal FU potentiation. In addition, pharmacokinetic studies have reported a significant variability of active folates in plasma after administration of standard-dose FA. Altogether, these observations favour high-dose FA administration to achieve high folate concentrations in plasma and thus to counteract the variability of the 1 FA concentrations required. With respect to the choice of FU-FA schedule, it appears from experimental data that increasing the duration of exposure to FA enhances FU-FA cytotoxicity, probably through an increased formation of reduced folate polyglutamate forms. Considering the S-phase specificity of FU cytotoxicity as well as its rapid elimination from plasma, a schedule of prolonged exposure to both FU and FA should be considered preferable. CONCLUSIONS: Results of the new FU-FA administration schedules such as the one consisting of a 2-hour FA administration followed by a combination of FU bolus and FU infusion, or the chronomodulated FU-FA infusion, open up promising approaches for improving the therapeutic index of FU-FA chemotherapy. Finally, future clinical studies should investigate tumoral parameters pharmacologically linked to FU-FA sensitivity such as pre-treatment TS and FPGS activities. Such tumoral investigations along with FU and FA pharmacokinetic investigations should provide a better understanding of inter-patient variability in response to FU-FA therapy and an optimal management of this chemotherapy regimen.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

A novel non-polyglutamable anti-folate, MX-68, inhibits the induction of experimental autoimmune uveitis in rats.

MX-68 is a novel antifolate which is chemically designed not to undergo intracellular polyglutamation thus preventing the development of adverse effects. The present study was carried out to examine both the in vitro and in vivo effects of MX-68 on experimental autoimmune uveitis (EAU) and to compare its effect on collagen-induced arthritis (CIA) in rats. EAU was induced by injecting Lewis rats with retinal S-antigen in complete Freund's adjuvant. Either MX-68 or methotrexate (MTX), which forms several polyglutamates intracellularly, was orally administered five days a week for three weeks beginning on the day of immunization. In vivo, both MX-68 and MTX significantly delayed the onset of EAU and inhibited the antibody response to S-antigen in a dose-dependent manner. High dose MX-68 (2.5 mg kg-1 day-1) completely abrogated the induction of EAU. No adverse effects were observed in either MX-68- or MTX-treated rats. However, the cessation of MX-68 administration after a period of three weeks resulted in the induction of EAU. In contrast, both MX-68 and MTX suppressed the severity of CIA without affecting the onset of the disease and inhibited anti-collagen antibody production in a dose-dependent fashion. Discontinuation of the drugs did not result in the recurrence of CIA. In vitro, both MX-68 and MTX significantly suppressed the proliferation of S-antigen- and Con A-stimulated lymph node cells obtained from immunized rats in a dose-dependent fashion. These data suggest that MX-68 may be useful for the treatment of autoimmune diseases including EAU and that the pathophysiology of EAU could be different from that of CIA.

2-Aminoadipic Acid↗

PubMed1987

The role of folylpolyglutamate synthetase and gamma-glutamyl hydrolase in altering cellular folyl- and antifolylpolyglutamates.

The metabolic products of methotrexate differ in hepatocytes and hepatoma cells; the primary products are 7-hydroxymethotrexate in hepatocytes and the polyglutamate derivatives in hepatoma cells. The rate of glutamylation is approximately 5-fold greater in hepatoma cells than hepatocytes under comparable conditions and the polyglutamate products in the hepatoma cells consist of longer chain length tri- and tetraglutamate derivatives, whereas the hepatocytes favor the diglutamate derivative. Extracts of cultured hepatocytes have approximately half the folylpolyglutamate synthetase and three times as much gamma-glutamyl hydrolase as the hepatoma cells. A further difference is that methionine inhibits glutamylation in hepatocytes whereas it can cause a marked stimulation (five- to seven-fold) in hepatoma cells. The rate of glutamylation of methotrexate in intact hepatoma cells is strongly dependent on the culture conditions and can be enhanced as much as five-fold in dividing cultures or by removing folates from the cells. These changes may be in part caused by the fact that the folylpolyglutamate synthetase is nearly doubled in both growing cultures and in folate lacking cells. The treatment of human liver in vivo or rodent hepatic cells in vivo and in vitro with methotrexate causes a reduction in the folate pools. Data presented in these studies show that the activity of gamma-glutamyl hydrolase in cells can be enhanced by prior exposure to methotrexate. Thus, methotrexate could cause a loss in cellular folylpolyglutamates by enhancing their rate of cleavage to folylmonoglutamates.

Animals↗

PubMed1992

Evidence for pteridine regulation of lead-mediated inhibition of uroporphyrinogen and heme formation in rat bone marrow.

Uroporphyrin I (URO I) accumulation has been reported in the bone marrow of rats exposed to lead, suggesting a sensitivity of uroporphyrinogen III cosynthase (COSYN) to this heavy metal. Furthermore, it has been reported that a polyglutamated folate derivative may serve as a coenzyme for the catalytic action of hepatic uroporphyrinogen III cosynthase. These findings raised the question of whether depletion of polyglutamated folate could enhance the susceptibility of bone marrow COSYN to lead and potentially interfere with the formation of heme. Nitrous oxide, an anesthetic agent capable of causing bone marrow tetrahydrofolate deficiency, depressed total bone marrow polyglutamated folate content by 42% with significant reductions in all three chain lengths (5-7) identified in the bone marrow during an exposure period of 7 days at 4 hr/day. Lead acetate (15 mg/kg) administered by ip injection at Days 0 and 2 during a 7-day exposure to nitrous oxide resulted in an 84% increase of bone marrow URO I content, which was markedly higher than the increases of 22 and 38% seen with sole administration of lead or nitrous oxide, respectively. The combination of agents also produced a 48% rise in COPRO I, a 39 and 43% decrease in COPRO III and protoporphyrin, respectively, and a 42% decline in the activity of microsomal 7-ethoxycoumarin O-deethylase, which is hemoprotein, cytochrome P-450 mediated. Heme oxygenase activity was not altered by nitrous oxide, lead, or their combination. These results suggest that bone marrow folate deficiency may render COSYN more sensitive to lead as characterized by increased uroporphyrin I and coproporphyrin I isomer content, decreased coproporphyrin III and protoporphyrin content, and depressed microsomal hemoprotein, cytochrome P-450-mediated drug-metabolizing capability.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1998

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase.

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-A resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Omega loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

Adenosine Triphosphate↗

PubMed1999

Recent advances in the understanding of the mechanism of membrane transport of folates and antifolates.

Transport of folates and antifolates in most cells is mediated by the reduced folate carrier (RFCI), an anion exchanging concentrative process, which is opposed by independent exit pump(s) that are directly coupled to energy metabolism. The balance of these processes governs the free intracellular folate level that is the substrate for folylpolyglutamate synthetase and determines the rate of metabolism of many folates and antifolates to polyglutamate derivatives. The RFCI has a high affinity for new-generation antifolates, but the relationship between transport and polyglutamylation for all these compounds has not, as yet, been defined. Analysis of mutations in RFCI indicate highly selective functional changes in binding affinity and/or carrier mobility, raising the possibilities that (1) enhanced transport and accumulation of natural folates can result in resistance to antifolate inhibitors of purine and pyrimidine synthesis by blocking their polyglutamylation to active derivatives, or by competition at the target enzyme site without substantial changes in antifolate transport, and (2) transport-related resistance to one antifolate might not produce comparable cross-resistance to another. Impaired export pump function, which results in enhanced accumulation of folates in cells and inhibition of antifolate polyglutamylation, represents another potential mechanism of resistance to new-generation antifolates. Folate receptor-mediated transport is another route of entry of folates and antifolates into cells, but its importance has not been well-defined. Finally, a low pH transport mechanism is present in a variety of mammalian cell lines. However, its relationship to RFCI and other low pH epithelial transporters and its ability to transport new-generation antifolates remain to be established.

Animals↗

PubMed1995

Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription.

L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H. (1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of 1,L5-CHO-folateH4 (natural diastereoisomer of 5-formyl-tetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constitutively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.

Amino Acid Sequence↗

PubMed2003

Early embryonic death of glutamate carboxypeptidase II (NAALADase) homozygous mutants.

Glutamate carboxypeptidase II (EC 3.4.17.21) catalyzes the hydrolysis (Km = 0.2 microM) of the neuropeptide N-acetylaspartylglutamate to yield N-acetylaspartate and glutamate and also serves as a high-affinity folate hydrolase in the gut, cleaving the polyglutamate chain to permit the absorption of folate. N-acetylaspartylglutamate is an agonist at the mGluR3 metabotropic receptor and a source of extracellular glutamate through hydrolysis by glutamate carboxypeptidase II. Given the important role of glutamate in brain development and function, we were interested in the effects of a null mutation of glutamate carboxypeptidase II that would potentiate the effects of N-acetylaspartylglutamate. The PGK-Neomycin cassette was inserted to delete exons 9 and 10, which we previously demonstrated encode for the zinc ligand domain essential for enzyme activity. Successful germline transmission was obtained from chimeras derived from embryonic stem cells with the targeted mutation of glutamate carboxypeptidase II. Homozygous null mutants did not survive beyond embryonic day 8. Folate supplementation of the heterozygous mothers did not rescue the homozygous embryos. Mice heterozygous for the null mutation appeared grossly normal and expressed both mutated and wild-type mRNA but the activity of glutamate carboxypeptidase II is comparable to the wild-type mice. The results indicate that the expression of glutamate carboxypeptidase II is upregulated when one allele is inactivated and that its activity is essential for early embryogenesis.

Aging↗

PubMed1995

Antifolates can potentiate topoisomerase II inhibitors in vitro and in vivo.

Antifolates have been shown to increase the DNA strand breaks produced by the topoisomerase inhibitor etoposide. PT523 is a potent new antifolate that cannot be polyglutamated. Human SCC-25 squamous carcinoma cells were exposed to methotrexate, trimetrexate or PT523 at a concentration of 5 microM for 24 h along with various concentrations of etoposide or novobiocin during the final 2 h. Isobologram analysis of the treatment combinations indicated that exposure of the cells to PT523/etoposide, methotrexate/etoposide, PT523/novobiocin, methotrexate/novobiocin and trimetrexate/novobiocin resulted in greater than additive cytotoxicity. DNA alkaline elution studies with the same drug combinations indicated that there were three- to four-fold increases in the radiation equivalent (rad equivalent) strand breaks in the cellular DNA with etoposide or novobiocin along with the antifolate compared with the topoisomerase II inhibitors alone. Tumor growth delay studies were carried out in the murine SCC VII squamous carcinoma. PT523 (0.5 mg/kg) and methotrexate (2 mg/kg) were administered by 7-day continuous infusion while trimetrexate (3.75 mg/kg) was administered intraperitoneally daily on days 7-9. Etoposide (10 mg/kg) and novobiocin (100 mg/kg) were administered intraperitoneally on alternate days (7, 9, 11). The combinations of PT523 with etoposide or novobiocin were significantly more effective than methotrexate and etoposide or novobiocin, producing tumor growth delays of 8.4 days and 6.9 days, respectively. Overall, the antifolate/topoisomerase II inhibitor treatment combinations produced tumor growth delays that were apparently additive to greater than additive.

Animals↗

PubMed1986

In vitro synthesis of alpha-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli.

Extracts of Escherichia coli contained an enzymatic activity which catalyzed the addition of L-glutamate to the alpha-carboxyl of various polyglutamate substrates, including folylpolyglutamates. Much of the enzyme activity was separated by DE52 chromatography and gel filtration from the enzyme which adds the first three glutamates in the biosynthesis of folylpolyglutamates, dihydrofolate synthetase-folylpolyglutamate synthetase. The two enzyme activities differed in many properties. Whereas dihydrofolate synthetase-folylpolyglutamate synthetase preferred pteroate or pteroylmonoglutamate substrates, the folylpoly-alpha-glutamate synthetase preparations effectively utilized tetrahydropteroylpolyglutamates, pteroylpolyglutamates, p-aminobenzoylpolyglutamates (pAB(Glu)n), and even a polyglutamate tripeptide. Several di- and triglutamyl peptides were inhibitory to folylpoly-alpha-glutamate synthetase activity, but not to dihydrofolate synthetase-folylpolyglutamate synthetase. Conversely, dihydropteroate noncompetitively inhibits the folylpolyglutamate synthetase reaction of the dihydrofolate synthetase-folylpolyglutamate synthetase protein, but did not inhibit the folylpoly-alpha-glutamate synthetase reaction. Potassium chloride was inhibitory to folylpoly-alpha-glutamate synthetase activity (as were other salts and several polyanions), in contrast to the absolute requirement of dihydrofolate synthetase-folylpolyglutamate synthetase activity for a monovalent cation such as K+. Incubation of a folylpoly-alpha-glutamate synthetase preparation with (6S)-tetrahydropteroyltri(gamma)glutamate generated products which after chemical cleavage to pAB(Glu)n were identical to those from growing E. coli, in high performance liquid chromatography retention times and in pattern of digestion by alpha-COOH bond-specific carboxypeptidase Y. High performance liquid chromatography and mass spectral analysis of the products of the in vitro reactions of folylpoly-alpha-glutamate synthetase with several substrates also demonstrated the addition of glutamate residues via alpha-COOH linkages. Thus, there appear to be two folylpolyglutamate synthetase activities in E. coli, dihydrofolate synthetase-folylpolyglutamate synthetase which adds the first three glutamate residues by gamma-COOH linkages and the folylpoly-alpha-glutamate synthetase activity which extends the folylpolyglutamate chain via gamma-COOH peptide bonds.

Carbon Radioisotopes↗

PubMed1970

Bacteriophage tail components. II. Dihydrofolate reductase in T4D bacteriophage.

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.

Aminopterin↗

PubMed2000

Interaction energy analyses of folate analog binding to human dihydrofolate reductase: contribution of the antifolate substructural regions to complex stability.

The three-dimensional structure of the human dihydrofolate reductase (DHFR), methotrexate tetrazole, and NADPH ternary complex was used to model the corresponding ternary complexes with methotrexate tetrazole replaced by methotrexate, methotrexate-polyglutamate with three glutamyl residues, and 5,10-deazaaminopterin, respectively. Each complex was solvated in a 60-angstrom cube of explicit water and subjected to structural minimization followed by interaction energy analyses. Interaction energy calculations were performed for the antifolate interaction with water, NADPH, the DHFR binding site residues, the entire DHFR protein, and the solvated NADPH:DHFR complex. These studies revealed that methotrexate-polyglutamate exhibited the most stable interactions and that approximately one half of antifolate:DHFR stability could be accounted for by the interaction of the antifolate with the binding site residues. The antifolate structures were also subdivided into heterocyclic, phenyl, and glutamyl substructural regions. Interaction energies were subsequently calculated for the interactions of the subregions with water, NADPH, the DHFR binding site residues, the DHFR protein, and the solvated NADPH:DHFR complex. The glutamyl substructural region showed the greatest contribution to overall antifolate binding stability due to its interaction with the DHFR protein. The heterocyclic and phenyl substructural regions generally showed much less stable interactions. These results suggest that the primary stabilizing factor of the antifolate interaction is the interaction of glutamyl with the DHFR protein. Additionally, interaction energy analyses were performed for specific groups of atoms within the substructural regions. These studies indicated that the stability of the glutamyl interaction is due to the interaction of glutamyl oxygen atoms with the DHFR protein. In the case of the methotrexate tetrazole complex, the tetrazole nitrogens also contribute significantly to the stability of the glutamyl interaction. The carbon atoms of the heterocyclic and phenyl groups both showed more stable interactions with NADPH than with water, while the nitrogen atoms showed more stable interactions with water than with NADPH. Collectively, these results indicate that the glutamyl region is the most important in antifolate binding stability.

Aminopterin↗

PubMed2002

The impact of phenylketonuria on folate metabolism.

Several reports indicate that biopterin and folate pathways may interact. We examined folate metabolism in PKU patients where hyperphenylalaninaemia leads to a likely excess of THB. We found an increase in total HPLC determined red cell folate in PKU (p=0.0422): specifically, there was an increase in total formyl-H(4)folate (p=0.0002) and H(4)folate (p< or =0.0001), and decrease in total 5-methyl-H(4)folate in PKU patients. At the level of individual oligo-gamma-glutamyl coenzymes, we found that formyl-H(4)folate polyglutamates were virtually all increased in PKU (p=0.0223, 0.0004, 0.0004, 0.0012, and 0.0008 for di-, tri-, tetra-, penta-, and deca-gamma-glutamyl formyl-H(4)folate coenzymes, respectively). Hcy levels did not differ between clinical groups, indicating that folate dependent-Hcy remethylation is not compromised as a consequence of an altered PKU folate disposition. In nature, pentaglutamyl folates are considered the metabolically favoured coenzymes (optimum K(m) for dependent enzymes). The presented data support this-we found that red cell pentaglutamates gave the best measure of metabolism; pentaglutamyl formyl-H(4)folate increased in PKU (p=0.0012) and related methenyls behaved similarly, while, pentaglutamyl 5-methyl-H(4)folate and pentaglutamyl H(4)folate decreased (p< or =0.0001 and 0.0265, respectively). Furthermore, pentaglutamates showed the best correlations between one-carbon oxidation states of folate, as well as with Hcy (p=0.0003 r=-0.54, 95% CI; -0.724 to -0.272). That PKU might influence folate metabolism in some way is unsurprising: patients with DHPR deficiency accumulate DHB and develop secondary folate deficiency-responsive only to reduced folates, while CSF levels of THB are significantly correlated to monoamines and red cell folate in depression. Further studies to confirm the present findings and to ascertain precisely what mechanism operates in PKU that impacts upon folate homeostasis so profoundly are required.

Biopterins↗