Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate

The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.     

Oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate by complex IV of rat mitochondria

We hypothesized that the unanticipated bioactivity of orally administered unnatural carbon-6 isomers, (6R)-5-formyltetrahydrofolate (5-HCO-THF) and (6S)-5,10-methenyltetrahydrofolate (5,10-CH-THF), in humans [Baggott, J. E., and Tamura, T. (1999) Biochim. Biophys. Acta 1472, 323-32] is explained by the rapid oxidation of (6S)-10-formyltetrahydrofolate (10-HCO-THF), which is produced by in vivo chemical processes from the above folates. An oxidation of 10-HCO-THF produces 10-formyldihydrofolate (10-HCO-DHF), which no longer has the asymmetric center at carbon-6 and is metabolized by aminoimidazole carboxamide ribotide (AICAR) transformylase forming bioactive dihydrofolate. Since cytochrome c (Fe(3+)) rapidly oxidizes both (6R)- and (6S)-10-HCO-THF [Baggott et al. (2001) Biochem. J. 354, 115-22], we investigated the metabolism of 10-HCO-THF by isolated rat liver mitochondria. We found that 10-HCO-THF supported the respiration of mitochondria without uncoupling ATP synthesis. The site of electron donation was identified as complex IV, which contains cytochrome c; the folate product was 10-HCO-DHF, and the reaction was saturable with respect to 10-HCO-THF. Both (6S)- (unnatural) and (6R)-10-HCO-THF supported the respiration of mitochondria, whereas (6S)-5-formyltetrahydrofolate (5-HCO-THF) was inactive. To our knowledge, this cytochrome c oxidation of 10-HCO-THF to 10-HCO-DHF in the mitochondrial intermembrane space represents a possible folate metabolic pathway previously unidentified and would explain the bioactivity of unnatural carbon-6 isomers, (6R)-5-HCO-THF and (6S)-5,10-CH-THF, in humans.     

pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates

The stability and eventual interconversion of nine mono-glutamate folates (5-methyl-tetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, 5,10-methylenetetrahydrofolate, dihydrofolate, 10-formylfolic acid, 10-formyltetrahydrofolate and folic acid) during the typical sample preparation steps (heat treatment for 10 min at 100 degrees C and incubation for 2 h at 37 degrees C) at different pH values have been investigated by LC-MS/MS. An LC-MS/MS method with isotopically labelled [(13)C(5)]5-methyltetrahydrofolate and [(13)C(5)] folic acid as internal standards has been developed with enhanced sensitivity using a Chromolith RP-18 column. 5-Methyltetrahydrofolate, folic acid and 10-formylfolic acid are relatively stable at different pHs (from 2 to 10) with and without heat treatment. Tetrahydrofolate shows instability at low pH. 5-Formyltetrahydrofolate and 5,10-methenyltetrahydrofolate can interconvert by changes in pH. Tetrahydrofolate and 5,10-methylenetetrahydrofolate can interconvert with formaldehyde or by changes in pH. Incubation at 37 degrees C for 2 h is much less aggressive for most folates as compared with heat treatment at 100 degrees C. At 37 degrees C most folates are stable at pH values between 4 and 8 except tetrahydrofolate and dihydrofolate, which are degraded at low pH. 10-Formyltetrahydrofolate and 5,10-methylenetetrahydrofolate cannot be quantified in the present method because these compounds are converted to 5,10-methenyltetrahydrofolate and tetrahydrofolate, respectively, in the acidic mobile phase. This study provides useful information for the analysis of folates in the future as well as for the interpretation of quantitative results from earlier work.     

Formyl-methenyl-methylenetetrahydrofolate synthetase from rabbit liver (combined). Evidence for a single site in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate.

The rabbit liver enzymes 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase, and 10-formyltetrahydrofolate synthetase have been purified to apparent homogeneity. Polyacrylamide gel electrophoresis patterns suggest a single protein is responsible for all three catalytic activities. The properties of the dehydrogenase and cyclohydrolase activities suggest that a single active site may catalyze these two reactions. This conclusion is based on spectral changes observed in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate, the similarity of dissociation constants determined from initial velocity studies for the two reactions, and the similarity of the pH-activity curves for the two reactions. NADP⁺ and NADPH lower the K_m for 5,10-methenyltetrahydrofolate 2- to 3-fold above pH 7 in the cyclohydrolase reaction but below pH 7 they act as partial inhibitors.     

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.     

Isolation, characterization, and structural organization of 10-formyltetrahydrofolate synthetase from spinach leaves

One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.     

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.     

Inhibition of mammalian thymidylate synthase by 10-formyltetrahydropteroylpolyglutamate

Reduced derivatives of 10-formylfolate have been evaluated as inhibitors of mammalian thymidylate synthase (EC 2.1.1.45) from H35 hepatoma cells. With 5,10-methylenetetrahydrofolylheptaglutamate as the substrate, 10-formyltetrahydrofolylmonoglutamate is a competitive inhibitor with a Ki of 2.4 microM, which is reduced to 0.1 microM for the heptaglutamate derivative. 10-Formyldihydrofolylmono- and -heptaglutamate are approximately threefold less inhibitory than the tetrahydro analog. The concentrations of 10-formyltetrahydrofolate and 10-formyldihydrofolate were measured in dividing hepatoma cells by a novel enzymatic assay and were found to be 5 microM and undetectable, respectively. These results suggest that the concentration of 10-formyltetrahydrofolate within the dividing cells has the potential to severely inhibit or modulate thymidylate biosynthesis.     

Bioactivity of orally administered unnatural isomers, [6R]-5-formyltetrahydrofolate and [6S]-5,10-methenyltetrahydrofolate, in humans

It has been assumed that humans cannot utilize 5,6,7,8-tetrahydrofolates with the unnatural configuration at carbon 6, since these folates are enzymatically and microbiologically inactive. We hypothesized that orally administered unnatural [6R]-5-formyltetrahydrofolate or [6S]-5,10-methenyltetrahydrofolate is bioactive in humans. Subjects were given independent oral doses of these unnatural folates and of a natural [6S]-5-formyltetrahydrofolate. Plasma, before and after the dose for 4 h, and 2 h urine were collected. Areas under the curve for the change in plasma folate concentrations were measured microbiologically and urinary folates were measured using HPLC. Based on findings of plasma and urinary folates, the unnatural folates were estimated to be 14-50% active as compared to [6S]-5-formyltetrahydrofolate. The major plasma and urinary folate was [6S]-5-methyltetrahydrofolate in all experiments. In urine, a [6S]-5-formyltetrahydrofolate peak was observed only after a [6S]-5-HCO-H4folate dose and peaks of unnatural [6S]-10-formyltetrahydrofolate and 5-formyltetrahydrofolate were identified after a [6R]-5-formyltetrahydrofolate dose. A possible pathway that explains our findings is discussed. This pathway includes the oxidation of the unnatural [6S]-10-formyltetrahydrofolate to 10-formyl-7,8-dihydrofolate which can be further metabolized by 5-amino-4-imidazolecarboxamide-ribotide transformylase producing dihydrofolate. Dihydrofolate can then be metabolized to [6S]-5-methyltetrahydrofolate by well-established metabolism.     

N10-Formyltetrahydrofolate is the formyl donor for glycinamide ribotide transformylase in Escherichia coli.

Glycinamide ribotide transformylase from Escherichia coli was obtained free of N5,N10-methenyltetrahydrofolate cyclohydrolase activity by DEAE-cellulose chromatography. In reaction mixtures containing this enzyme preparation in potassium maleate buffer, pH 7.2, no detectable interconversion of N5,N10-methenyltetrahydrofolate occurred. Upon addition of glycinamide ribotide, N-formylglycinamide ribotide was formed when N10-formyltetrahydrofolate was present; no formylation occurred in the presence of N5,N10-methenyltetrahydrofolate. A method for the synthesis and purification of glycinamide ribotide is presented.     

Inhibition of 5,10-methenyltetrahydrofolate synthetase

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.     

Isolation and sequencing of the cDNA coding for spinach 10-formyltetrahydrofolate synthetase. Comparisons with the yeast, mammalian, and bacterial proteins.

The one-carbon metabolism enzymes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) can be found on a single trifunctional protein in the eukaryotes examined. The one exception is in spinach leaves where 10-formyltetrahydrofolate synthetase is monofunctional (Nour, J. M., and Rabinowitz, J. C. (1991) J. Biol. Chem. 266, 18363-18369). In the prokaryotes examined, 10-formyltetrahydrofolate synthetase is either absent or is monofunctional. A cDNA clone encoding spinach leaf 10-formyltetrahydrofolate synthetase was isolated through the use of antibodies to the purified enzyme. This clone had an open reading frame of 1914 base pairs and encoded for a protein containing 636 amino acids with a calculated M(r) of 67,727. The percentage identity between spinach 10-formyltetrahydrofolate synthetase and the synthetase domains in the four trifunctional eukaryotic enzymes and the two monofunctional prokaryotic enzymes that have been cloned and sequenced was: 64.9% human, 63.8% rat, 55.6% yeast cytoplasm, 53.8% yeast mitochondria, 47.8% Clostridium acidi-urici, and 47.9% Clostridium thermoaceticum. Clearly the spinach monofunctional protein had greatest homology with the mammalian proteins. The spinach protein is longer than the two other monofunctional prokaryotic proteins. Possible reasons for this are presented. The codon usage and the putative translation initiation sites are examined and compared with other spinach proteins.     

Bacterial conversion of folinic acid is required for antifolate resistance

Antifolates, which are among the first antimicrobial agents invented, inhibit cell growth by creating an intracellular state of folate deficiency. Clinical resistance to antifolates has been mainly attributed to mutations that alter structure or expression of enzymes involved in de novo folate synthesis. We identified a Mycobacterium smegmatis mutant, named FUEL (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. Chemical complementation indicated that FUEL is unable to metabolize folinic acid (also known as leucovorin or 5-formyltetrahydrofolate), whose metabolic function remains unknown. Targeted mutagenesis, genetic complementation, and biochemical studies showed that FUEL lacks 5,10-methenyltetrahydrofolate synthase (MTHFS; also called 5-formyltetrahydrofolate cyclo-ligase; EC 6.3.3.2) activity responsible for the only ATP-dependent, irreversible conversion of folinic acid to 5,10-methenyltetrahydrofolate. In trans expression of active MTHFS proteins from bacteria or human restored both antifolate resistance and folinic acid utilization to FUEL. Absence of MTHFS resulted in marked cellular accumulation of polyglutamylated species of folinic acid. Importantly, MTHFS also affected M. smegmatis utilization of monoglutamylated 5-methyltetrahydrofolate exogenously added to the medium. Likewise, Escherichia coli mutants lacking MTHFS became susceptible to antifolates. These results indicate that folinic acid conversion by MTHFS is required for bacterial intrinsic antifolate resistance and folate homeostatic control. This novel mechanism of antimicrobial antifolate resistance might be targeted to sensitize bacterial pathogens to classical antifolates.     

Facile new synthesis and purification of 5,10-methenyltetrahydrofolate from folic acid

A facile new synthesis of tetrahydrofolate is reported starting with folic acid and reducing with sodium cyanoborohydride in glacial acetic acid. Conversion to 5,10-methenyltetrahydrofolate is effected by heating with formic acid in acetic acid, followed by purification on phosphocellulose at acid pH.     

Fermentation of Fructose and Synthesis of Acetate from Carbon Dioxide by Clostridium formicoaceticum

Clostridium formicoaceticum ferments fructose labeled with (14)C in carbon 1, 4, 5, or 6 via the Embden Meyerhof pathway. In fermentations of fructose in the presence of (14)CO(2), acetate is formed labeled equally in both carbons. Extracts convert the methyl groups of 5-methyltetrahydrofolate and methyl-B(12) to the methyl group of acetate in the presence of pyruvate. Formate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, and 5,10-methylenetetrahydrofolate reductase are present in extracts of C. formicoaceticum. These enzymes are needed for the conversion of CO(2) to 5-methyltetrahydrofolate. It is proposed that acetate is totally synthesized from CO(2) via the reactions catalyzed by the enzymes listed above and that 5-methyltetra-hydrofolate and a methylcorrinoid are intermediates in this synthesis.     

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

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

Reconstitution of Escherichia coli DNA photolyase with various folate derivatives

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.     

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.     

Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis>

5,10-Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate coupled to the hydrolysis of ATP. A co-crystal structure of MTHFS bound to its substrates has been published (Chen et al., Proteins 56:839-843, 2005) that provides insights into the mechanism of this reaction. To further investigate this mechanism, we have replaced the arginine at position 115 and the lysine at position 120 with alanine (R115A and K120A, respectively). Circular dichroism spectra for both mutants are consistent with folded proteins. R115A shows no activity, suggesting that R115 plays a critical role in the activity of the enzyme. The K120A mutation increases the Michaelis constant (K(m)) for ATP from 76 to 1,200 muM and the K(m) for 5-formylTHF from 2.5 to 7.1 muM. The weaker binding of substrates by K120A may be due to movement of a loop consisting of residues 117 though 120, which makes several hydrogen bonds to ATP and may be held in position by K120.     

Human red blood cell-mediated metabolism of leucovorin [(R,S)5-formyltetrahydrofolate

The ability of human blood in vitro, and partially purified red blood cells, to metabolize leucovorin, or 5-formyltetrahydrofolate, has been examined. A radioenzymatic assay based upon entrapment of 5,10-methylenetetrahydrofolate, and other reduced folates after cycling to this form, into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxyuridine-5'-monophosphate was used to estimate reduced folate metabolites. Incubation of whole blood samples with (R,S)5-formyltetrahydrofolate resulted in a time- and concentration-dependent extracellular accumulation of the reduced folates, 5-methyltetrahydrofolate, tetrahydrofolate, 10-formyltetrahydrofolate, and 5,10-methylenetetrahydrofolate. While accumulation with time was nonlinear, the tetrahydrofolate pool showed the greatest overall increase in concentration. 5-Methyltetrahydrofolate, which was the only reduced folate detected in plasma prior to introduction of (R,S)5-formyltetrahydrofolate, accumulated more slowly than tetrahydrofolate. 10-Formyltetrahydrofolate and 5,10-methylenetetrahydrofolate accumulated even more slowly but exhibited nonlinear kinetic patterns similar to those of tetrahydrofolate and 5-methyltetrahydrofolate. When blood cells were removed by centrifugation, a complete loss of metabolic activity was observed. Exposure of purified red blood cells to (R,S)5-formyltetrahydrofolate resulted in accumulation of extracellular reduced folates that was similar to that in whole blood samples while partially purified white blood cells exhibited little activity. Metabolism of the (S) diastereomer of 5-formyltetrahydrofolate accounted for essentially all of the observed extracellular accumulation of reduced folates. We propose that red blood cell-mediated metabolism of 5-formyltetrahydrofolate could, in part at least, account for reduced folate accumulation in plasma when leucovorin is administered to humans.