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PubMed1995

Antitumour effects of pure diastereoisomers of 5-formyltetrahydrofolate in hepatic transplants of a rodent colon carcinoma model.

The effects of the two diastereoisomers of 5-formyltetrahydrofolate on tumour growth, thymidylate synthase (TS, EC 2.1.1.45) levels, and potentiation of 5-fluorouracil cytotoxicity were studied in an in vivo rat colon carcinoma model, transplanted to liver. The animals were randomized into eight groups, treated with daily i.v. tail vein injections of racemic (d,l)-5-formyltetrahydrofolate (5-CHO-FH4), 15 mg/kg, (1)-5-CHO-FH4 7.5 mg/kg, and (d)-5-CHO-FH4 7.5 mg/kg, 5-fluorouracil (FUra) 30 mg/kg, (d,l)-5-CHO-FH4 15 mg/kg+FUra 30 mg/kg, (l) 5-CHO-FH4 7.5 mg/kg+FUra 30 mg/kg, and (d)-5-CHO-FH4 7.5 mg/kg+FUra 30 mg/kg, and a sham-treated control group. The average tumour size of the groups was equal at the start of treatment. After six days' treatment the average tumour sizes were at laparotomy 3.3 +/- 1.0 g in the (d/l)-5-CHO-FH4 treated group, compared to 2.0 +/- 0.1 g in the FUra treated group and 7.1 +/- 3.1 g in the controls. Natural (l)-5-CHO-FH4 promoted tumour growth (average tumour weight 10.8 +/- 4.0 g), whereas the unnatural (d)-5-CHO-FH4 alone retarded it (average tumour weight 1.2 +/- 0.40 g). (l)-5-CHO-FH4 induced a significant increase in tumour tissue TS levels by [3H]FdUMP radioligand assay (27.5 +/- 8.4 pmol/g tumour tissue) compared to controls (16.8 +/- 6.1 pmol/g tumour tissue). Increases in 5,10-methylenetetrahydrofolate and tetrahydrofolate occurred with FUra alone, with a further statistically significant increase in both folates with the addition of (d)-5-CHO-FH4 to FUra.

Animals↗
PubMed1984

Enzymatic activation of 5-formyltetrahydrofolate via conversion to 5, 10-methenyltetrahydrofolate.

The ATP-dependent conversion of 5-formyltetrahydrofolate (folinate) to the 5,10-methenyl derivative, catalyzed by 5,10-methenyltetrahydrofolate synthetase (EC 6.3.3.2), is of considerable importance in cancer chemotherapy, since it provides the basis for the administration of folinate to counteract the deleterious effects of high-dose Methotrexate regimens. Methenyltetrahydrofote synthetase has been purified 10,000-fold from L. casei using sequential affinity chromatography on immobilized folinate and ATP. The monomeric enzyme is homogeneous upon SDS-polyacrylamide gel electrophoresis, has a molecular weight of 23,000 (confirmed by gel filtration), and contains a single cysteine residue. The turnover number is ca. 250 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively; the equilibrium constant is 0.7-1.0 mM. Methotrexate, 5-methyltetrahydrofolate, and folate are not inhibitory. The mechanism for the reaction is proposed to involve phosphorylation of the formyl group to create an enol phosphate; subsequent attack on the methenyl carbon by N-10 would generate a tetrahedral intermediate, with release of the phosphate providing the driving force for ring closure.

Animals↗
PubMed1986

Nucleotide stereochemistry in the formyltetrahydrofolate synthetase reaction.

In a recent study, we have shown that N10-formyltetrahydrofolate synthetase prefers (Sp)-MgATP beta S over the Rp isomer in the forward reaction. In this report the stereochemistry of ATP beta S produced from prochiral ADP beta S in the reverse reaction was determined. The ATP beta S product was purified and tested as a substrate for hexokinase (preference for the Rp isomer), adenylate kinase (preference for the Sp isomer) and N10-formyltetrahydrofolate synthetase. A comparison of kinetic constants for the product and the authentic Sp and Rp isomers shows that the product is the Sp diastereomer. 31P NMR was also used to identify the product as (Sp)-ATP beta S.

Adenosine Triphosphate↗
PubMed1991

Preparation of a chiral matrix. Resolution of (6R,S)-N5-formyltetrahydrofolate.

Combination chemotherapy involving (6R,S)-N5-formyltetrahydrofolate and 5-fluorouracil has raised considerable speculation concerning the effects of the unnatural (6R) diastereomer. The inability to obtain quantities of the individual diastereomers has greatly limited work in this area. Commercially available chiral columns, suitable for diastereomer analysis, are inadequate for preparative work. We report here on the use of epoxide-activated media in the construction of a bovine serum albumin-based high-performance liquid chromatography matrix capable of resolving the diastereomers of (6R,S)-N5-formyltetrahydrofolate in milligram quantities. Similar columns based upon alternative protein matrices may prove useful for the resolution of additional materials.

Adenocarcinoma↗
PubMed1989

Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction.

N10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the "formate kinase" activity was part of the catalytic center of N10-formyltetrahydrofolate synthetase was shown by demonstrating coordinate inactivation of the "kinase" and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar Ki values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10(-4) and 4 x 10(-6) of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a "burst" of ADP formation (6.4 s-1) was observed that is considerably faster than the steady-state rate (0.024 s-1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of N10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a "burst" of product formation. The rate of this burst phase at 5 degrees C occurs with a rate constant of 18 s-1 compared to 14 s-1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normal catalytic pathway. Positional isotope exchange experiments using [beta,gamma-18O]ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the 18O label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P beta oxygens of the product ADP.

Adenosine Diphosphate↗
PubMed1989

In vitro conversion of formate to serine: effect of tetrahydropteroylpolyglutamates and serine hydroxymethyltransferase on the rate of 10-formyltetrahydrofolate synthetase.

Serine hydroxymethyltransferase and C1-tetrahydrofolate synthase catalyze four reactions which convert formate and glycine to serine. The one-carbon carrier in these reactions if tetrahydropteroylglutamate which is regenerated in the coupled reaction and thus can be used in catalytic concentrations with respect to serine synthesis. The rate of serine synthesis is followed by the oxidation of NADPH during reduction of the intermediate 5,10-methenyltetrahydropteroylglutamate. Km values for the substrates of cytosolic serine hydroxymethyltransferase and the 10-formyltetrahydrofolate synthetase activity of the trifunctional enzyme C1-tetrahydrofolate synthase were determined. This included the values for the polyglutamate forms of tetrahydropteroylglutamate containing from one to six glutamate residues. The results suggest that the synthetase active site binds the polyglutamate forms of the coenzyme synergistically with respect to formate and ATP. Using saturating levels of all substrates, the kcat values for the serine hydroxymethyltransferase and 10-formyltetrahydrofolate synthetase activities were also determined. The synthetase reaction is the rate-determining step in the conversion of formate to serine. The effect of glutamate chain length and the concentration of serine hydroxymethyltransferase were studied with respect to the rate of serine formation. Tetrahydropteroylmonoglutamate gave slower than expected rates which is attributed to its inhibition of the reduction of the intermediate 5,10-methenyltetrahydropteroylglutamate. This inhibition was not a factor with the di- through hexaglutamate forms of the coenzyme. The addition of an excess of serine hydroxymethyltransferase was predicted to lower the rate of the formation of serine by lowering the concentration of free coenzyme in the assay. However, activation of the rate was observed which was at least 2-fold greater than the predicted rate. This increase in predicted rate appears to result from an interaction between C1-tetrahydrofolate synthase and serine hydroxymethyltransferase. The in vivo concentrations of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in rabbit liver were determined.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2002

Cloning and characterization of mitochondrial 5-formyltetrahydrofolate cycloligase from higher plants.

5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria. 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria. Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides. These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA. The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms. Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence. Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms. Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process. Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria.

Amino Acid Sequence↗
PubMed1989

Lack of interference by the unnatural isomer of 5-formyltetrahydrofolate with the effects of the natural isomer in leucovorin preparations.

Leucovorin, a mixture of the natural (6S) and unnatural (6R) diastereomers of 5-formyltetrahydrofolate, is administered clinically to enhance the antitumor activity of 5-fluorouracil. Because the 6R isomer persists at high concentrations in plasma for prolonged periods after iv leucovorin administration (J Clin Oncol 4:685-696, 1986), we have examined it to ascertain whether the 6R isomer could interfere with the cellular effects of the 6S isomer. The 6R compound had a poorer uptake into human CCRF-CEM lymphoblastic cells than the 6S compound, but the 6R compound could competitively inhibit the uptake of the natural isomer as determined in defined buffers. However, the 6R compound failed to interfere with cell growth support and enhancement of 5-fluorouracil cytotoxicity by the 6S isomer in CCRF-CEM cells in tissue culture experiments at concentrations up to 1 mM. Thus, the unnatural isomer of 5-formyltetrahydrofolate present in leucovorin preparations seems unlikely to have clinically relevant consequences.

Cell Division↗
PubMed1997

Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves.

Leaf extracts of 14-d-old pea (Pisum sativum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,10-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,10-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1% of total leaf folate and less that 1% of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. Immunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). Two mitochondrial protein bands (subunit Mr = 40,000 and 44,000) cross-reacted with SYN-Ab. Immunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fell from 5 to 1 [mu]mol min-1 mg-1 protein and less SYN-Ab cross-reacting protein was detected. The data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 10-formyltetrahydrofolate are extra-mitochondrial.

Journal Article↗
PubMed1982

A comparison of tetrahydrofolate and 5-formyltetrahydrofolate in correcting the impairment of thymidine synthesis in pernicious anaemia.

5-formyltetrahydrofolate and tetrahydrofolate were added to marrow cells from patients with untreated pernicious anaemia at 1, 5 and 50 nmol doses in the deoxyuridine suppression test. At all 3 dose levels formyltetrahydrofolate was significantly more effective in correcting the defect of thymidine synthesis in pernicious anaemia, than tetrahydrofolate. The data suggest that formylation of tetrahydrofolate is necessary for its normal utilization.

Anemia, Pernicious↗
PubMed1964

REPRESSION BY ADENINE OF THE FORMYLTETRAHYDROFOLATE SYNTHETASE IN AN ANTIFOLIC-RESISTANT MUTANT OF STREPTOCOCCUS FAECALIS.

Albrecht, Alberta M. (Sloan-Kettering Institute for Cancer Research, New York, N.Y.), and Dorris J. Hutchison. Repression by adenine of the formyltetrahydrofolate synthetase in an antifolic-resistant mutant of Streptococcus faecalis. J. Bacteriol. 87:792-798. 1964.-In an amethopterin-resistant mutant of Streptococcus faecalis ATCC 8043 under cultivation conditions requiring purine synthesis de novo, both the dihydrofolate reductase and the formyltetrahydrofolate synthetase were formed as constant fractions of the total protein synthesized during the exponential phase of growth. When excess adenine was added to the medium, the rate of formation of the synthetase was markedly decreased, i.e., repressed. Under these latter conditions, the synthesis of the reductase proceeded at a rate equal to that observed in the absence of adenine. The repressibility of the synthetase by adenine was demonstrated also by the decrease in rate of synthetase formation upon the addition of adenine to a culture actively synthesizing this enzyme. Guanine and hypoxanthine, like adenine, also repressed the synthetase; exogenous xanthine was less effective. Neither of the pyrimidines, thymine and uracil, at approximately 1 mug/ml, interfered with synthesis of the two enzymes.

Adenine↗
PubMed1971

Folates in plasma and bile of man after feeding folic acid--3H and 5-formyltetrahydrofolate (folinic acid).

During the 1st hr after feeding folic acid-(3)H ((3)H-PteGlu) to fasting human volunteers, plasma S. faecalis and (3)H activity were elevated to an equivalent degree, whereas after this, the (3)H activity exceeded S. faecalis activity, which suggests gradual conversion of folic acid-(3)H to methyltetrahydrofolate-(3)H (5-CH(3)H(4) PteGlu). The increase of L. casei activity exceeded the increase of S. faecalis and (3)H activity, which is consistent with flushing of endogenous methyltetrahydrofolate from the tissues by the administered folic acid-(3)H. Feeding of 5-formyltetrahydrofolate (+/-5CHOH(4)PteGlu) produced a large increase of plasma L. casei activity and only a slight increase of S. faecalis and P. cerevisiae activity, which is consistent with very rapid conversion of folinic acid to methyltetrahydrofolate. Bile folate concentration determined microbiologically was 2.3-9.8 times plasma folate. 40-80% of the bile folate was S. faecalis-active and 20-35% P. cerevisiae-active. Chromatography of bile folates on TEAE-cellulose showed several folates including four tentatively identified as 10-formyltetrahydrofolate (10-CHO-H(4)PteGlu), 10-formylfolate (10-CHO-PteGlu), and/or 10-formyldihydrofolate (10-CHOH(2)PteGlu), methyltetrahydrofolate, and possibly a triglutamate folate. After folate ingestion bile folate concentration increased rapidly. The distribution of bile folates measured by microbiological assay was similar after either folic or folinic acid feeding. Most of the (3)H label of folic acid-(3)H appeared in the biological folates of bile rather than in the folic acid fraction, which shows that the administered folic acid was rapidly transformed to other folates. Folate polyglutamate deconjugating enzyme activity was found to be much less than in serum. Polyglutamates of the type found in yeast were not found in bile. It is suggested that biliary folate may reflect the hepatic intracellular oligoglutamate folate pool rather than the folate as it appears in the hepatic portal blood.

Bile↗
PubMed1971

Transport characteristics of folates in cerebrospinal fluid; a study utilizing doubly labeled 5-methyltetrahydrofolate and 5-formyltetrahydrofolate.

Serum disappearance curves in dogs after the intravenous injection of radioactive methotrexate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and folic acid followed first-order kinetics with half-disappearance times ranging from 1.3 to 9 hr respectively. Equilibration of spinal fluid tritium levels with those in serum was rapid (3.0 hr) for both of the reduced folates but was not observed at any time after folic acid and methotrexate. The only radioactive folate identified in the spinal fluid after intravenous injection of either 5-formyltetrahydrofolate or folic acid, as well as 5-methyltetrahydrofolate was 5-methyltetrahydrofolate. These findings indicated that 5-methyltetrahydrofolate was taken up preferentially into the spinal fluid and that the other folate congeners were converted to this compound before uptake. Diphenylhydantoin administration did not alter the uptake of 5-methyltetrahydrofolate into the spinal fluid but was associated with reduced renal excretion of this compound.

Anemia, Macrocytic↗
PubMed1992

Depletion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate by methotrexate in cultured hepatoma cells.

The effect of the inhibition of dihydrofolate reductase by methotrexate on the cellular folates involved in de novo purine and thymidylate biosynthesis has been measured in H35 hepatoma cells grown in 4 microM folic acid or 20 nM folinic acid. The major cellular folate species in cells from medium with folate or folinate is 10-formyltetrahydrofolate (approximately 5 microM), with lesser amounts of 5,10-methylenetetrahydrofolate and tetrahydrofolate. Cultures were exposed to a pulse dose of methotrexate, resulting in the accumulation of nearly exclusively methotrexate polyglutamates (predominantly Glu3, Glu4, and Glu5), or a continuous exposure to the poorly glutamylated analog threo-4-fluoromethotrexate, resulting in 93% intracellular monoglutamate. At 4 hr and 18 hr after exposure to either compound there was extensive depletion of the reduced folate coenzymes, which generally corresponded to the extent of inhibition of glycine and deoxyuridine incorporation. This was accompanied by an increase of the cellular dihydrofolate and 10-formyldihydrofolate. In the H35 cells the effect of methotrexate polyglutamates on the reduced folate coenzyme pools was restricted to dividing cultures, because the reduced folate coenzymes were not depleted in confluent cultures. The results demonstrate that the methotrexate and methotrexate polyglutamates that initially accumulate within dividing H35 cells readily inhibit dihydrofolate reductase but are not adequate to inhibit thymidylate synthase and prevent the depletion of reduced folate coenzymes. Thus, inhibition of de novo glycine and deoxyuridine incorporation into DNA as a result of dihydrofolate reductase inhibitors appears to be closely related to a reduction in the intracellular concentration of 10-formyltetrahydrofolate and 5,10-methylenetetrahydrofolate, the respective folate coenzymes for de novo purine and thymidylate synthesis.

Animals↗
PubMed1987

10-Formyltetrahydrofolate synthetase. Evidence for a conformational change in the enzyme upon binding of tetrahydropteroylpolyglutamates.

The 10-formyltetrahydrofolate synthetase domain of the trifunctional enzyme C1-tetrahydrofolate synthase appears to undergo a conformational change in the presence of tetrahydropteroylpolyglutamates, MgATP, and ammonium ion. The binding of these ligands increases the denaturation temperature of the enzyme by 12 degrees C, abolishes the cold lability of the enzyme, and alters its susceptibility to digestion by chymotrypsin. The results suggest that a conformational change is dependent upon binding of the third glutamate residue of tetrahydropteroylpolyglutamates and the beta-phosphoryl group of MgATP. The Km values for MgATP and formate are lowered 3.6- and 520-fold, respectively, when tetrahydropteroyltriglutamate is used as the substrate in place of tetrahydropteroylmonoglutamate. A sensitive coupled assay involving C1-tetrahydrofolate synthase and serine hydroxymethyltransferase was developed to determine the activity of 10-formyltetrahydrofolate synthetase. The assay gives linear rates with the tetrahydropteroylpolyglutamates as substrates but not with the monoglutamate form.

Adenosine Triphosphate↗
PubMed1980

Formyl-methenyl-methylenetetrahydrofolate synthetase(combined) from yeast. Biochemical characterization of the protein from an ade3 mutant lacking the formyltetrahydrofolate synthetase function.

A protein from Saccharomyces cerevisiae mutant ade3-1050, a formyltetrahydrofolate synthetase-deficient mutant, has been purified to apparent homogeneity. The purified mutant enzyme shows both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, but lacks formyltetrahydrofolate synthetase activity. The biochemical characterization of the mutant protein described in this paper is consistent with genetic data which indicate that the 1050 mutation is a point mutation at the ade3 locus of chromosome VII of S. cerevisiae. The molecular weight of the native mutant protein (Mr = 227,000 by exclusion chromatography), as well as the number and size of its subunits are exactly the same as those of the trifunctional wild type enzyme. In addition, both proteins have the same sedimentation behavior in a glycerol density gradient (s20,w = 9.4 S), and their activities and structures are equally affected by exposure to mild tryptic degradation. ATP protects both enzymes from tryptic degradation, but NADP+ does not. Some of the kinetic properties of the activities of both enzymes were also determined and were essentially similar. Although both enzymes require the presence of metals for maximal synthetase and dehydrogenase activities, metals are not necessary to maintain their structures intact.

Aminohydrolases↗
PubMed1999

A noncatalytic tetrahydrofolate tight binding site is on the small domain of 10-formyltetrahydrofolate dehydrogenase.

10-Formyltetrahydrofolate dehydrogenase has previously been identified as a tight binding protein of the polyglutamate forms of tetrahydrofolate (R. J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982). Each subunit contains two independently folded domains connected by a linking peptide. By using the stable substrate and product analogs 10-formyl 5,8-dideazafolate and 5, 8-dideazafolate, respectively, we have determined that the tight binding folate site is separate from the catalytic site and that it is located on the N-terminal domain of the protein. This was achieved by cross-linking 10-formyl 5,8-dideazafolate to the dehydrogenase through the carboxyl group of the substrate analog. The cross-linked substrate analog was converted to the cross-linked product complex by adding either NADP+ or 2-mercaptoethanol, proving that the 10-formyl 5,8-dideazafolate was bound at the active site. With the active site cross-linked to 5,8-dideazafolate and not available for binding, the enzyme still bound 5, 8-dideazafolate-[3H]tetraglutamate tightly but noncovalently. Separation of the large and small domains by limited proteolysis showed that the tightly bound 5,8-dideazafolate-[3H]tetraglutamate was located on the small domain. The location of the cross-linked 10-formyl 5,8-dideazafolate at the active site was determined by amino acid sequencing of an isolated tryptic peptide.

Amino Acid Sequence↗