Oxidative and reductive cleavage of folates--a critical appraisal.

A critical analysis has been made of the oxidative and reductive techniques employedfor cleavage of the C⁹-N¹⁰ bond of folic acid and its derivaatives. The assumption has previously been made that these cleavage reactions reduce folates to a common family of p-aminobenzoylglutamate derivatives varying only in the lengths of γ-polyglutamyl peptide side chains which are readily subjected to quantitative and qualitative analysis. This assumption is incorrect. Oxidation by potassium permanganate effectively cleaved folic acid, dihydrofolic acid, tetrahydrofolic acid, and 5-formyltetrahydrofolic acid to yield p-aminobenzoylglutamate. 5-Methyltetrahydrofolic acid was merely oxidized to 5-methyldihydrofolic acid while 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were oxidized to 10-formylfolate which was stable to further attack. Of all the folate derivatives tested only folic acid and dihydrofolic acid were cleaved to p-aminobenzoylglutamate by the zinc-hydrochloric acid reduction method. Both tetrahydrofolic acid and 5-methyltetrahydrofolic acid were stable under fully reducing conditions. 5,10-Methenyl-,10-formyl-, and 5-formyltetrahydrofolic acid yielded N-methyl-p-aminobenzoylglutamate. It is evident, therefore, that not only is the dominant mammalian tissue folate derivative, 5-methyltetrahydrofolate, resistant to cleavage by either method, but that a common family of p-aminobenzoylglutamate derivatives is not the end product of those folate compounds that are susceptible. While this may not invalidate the reports of the relative polyglutamate chain lengths of tissue folates such data should be regarded with some caution.     

Methenyltetrahydrofolate synthetase is a high-affinity catecholamine-binding protein

Recombinant mouse 5,10-methenyltetrahydrofolate synthetase (MTHFS) was expressed in Escherichia coli and shown to co-purify with a chromophore that had a lambda(max) at 320nm. The chromophore remained bound to MTHFS during extensive dialysis, but dissociated from MTHFS when its substrate, 5-formyltetrahydrofolate, was bound. The chromophore was identified as an oxidized catecholamine by mass spectrometry and absorption spectroscopy. Purified recombinant mouse MTHFS and rabbit liver MTHFS proteins were shown to bind oxidized N-acetyldopamine (NADA) tightly. The addition of NADA to cell culture medium accelerated markedly folate turnover and decreased both folate accumulation and total cellular folate concentrations in MCF-7 cells. Expression of the MTHFS cDNA in MCF-7 cells increased the concentration of NADA required to deplete cellular folate. The results of this study are the first to identify a link between catecholamines and one-carbon metabolism and demonstrate that NADA accelerates folate turnover and impairs cellular folate accumulation in MCF-7 cells.     

Binding of radiolabeled folate and 5-methyltetrahydrofolate to cow's milk folate binding protein at pH 7.4 and 5.0. Relationship to concentration and polymerization equilibrium of the purified protein

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 M) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.     

In Vitro Studies of 5,10-Methylenetetrahydrofolate Reductase: Inhibition by Folate Derivatives, Folate Antagonists, and Monoamine Derivatives

Abstract: Folate monoglutamates and folate antagonists have an inhibitory action on the activity of 5, 10-methylenetetrahydrofolate reductase in rat brain. The type of inhibition was studied for dihydrofolic acid using the Lineweaver–Burk transformation. Some of the monoamine alkaloids, the in vitro products of 5, 10-methylenetetrahydrofolate reductase, have either a stimulatory or inhibitory effect on the enzyme activity.     

Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.     

Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a spartan strategy for a ping-pong reaction

Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (betaalpha)(8) barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH(3)-H(4)folate have now been determined at resolutions of 1.95 and 1.85 A, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH(3)-H(4)folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta2-alpha2 (L2), beta3-alpha3 (L3), and beta4-alpha4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.     

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.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: roles for aspartate 120 and glutamate 28

The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate). The X-ray crystal structure of the enzyme has revealed the amino acids at the flavin active site that are likely to be relevant to catalysis. Here, we have focused on two conserved residues, Asp 120 and Glu 28. The presence of an acidic residue (Asp 120) near the N1-C2=O position of the flavin distinguishes MTHFR from all other known flavin oxidoreductases and suggests an important function for this residue in modulating the flavin reactivity. Modeling of the CH(3)-H(4)folate product into the enzyme active site also suggests roles for Asp 120 in binding of folate and in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. In the NADH-menadione oxidoreductase assay and in the isolated reductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential. Compared to the wild-type enzyme, the mutant showed 150-fold decreased activity in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction and in the oxidative half-reaction involving CH(2)-H(4)folate, but the apparent K(d) for CH(2)-H(4)folate was relatively unchanged. Our results support a role for Asp 120 in catalysis of folate reduction and perhaps in stabilization of the 5-iminium cation. By analogy to thymidylate synthase, which also uses CH(2)-H(4)folate as a substrate, Glu 28 may serve directly or via water as a general acid catalyst to aid in 5-iminium cation formation. Consistent with this role, the Glu28Gln mutant was unable to catalyze the reduction of CH(2)-H(4)folate and was inactive in the physiological oxidoreductase reaction. The mutant enzyme was able to bind CH(3)-H(4)folate, but reduction of the FAD cofactor was not observed. In the NADH-menadione oxidoreductase assay, the mutant demonstrated a 240-fold decrease in activity.     

Solid-phase extraction-electrospray ionization mass spectrometry for the quantification of folate in human plasma or serum

The measurement of 5-methyltetrahydrofolic acid (5 MT) blood levels is one of several factors used to diagnose folate deficiency in humans. 5 can be selectively purified from either human plasma or human serum via solid-phase extraction procedures and specifically detected and quantified in the extracts with liquid chromatography/isotope-dilution electrospray-ionization mass spectrometry. Two different, yet complementary, solid-phase extraction-liquid chromatography/mass spectrometry methods have been developed and applied to the quantification of 5 MT from such extracts. One method utilizes the high-affinity folate-binding protein from cow's milk coupled with multiple-reaction-monitoring-mode tandem mass spectrometry while the other method utilizes reversed-phase C(18) extraction followed by selected-ion-monitoring-mode mass spectrometry. The accuracy of each method is assessed through a comparative determination of 5 MT levels in homogenous plasma and serum pools. Additionally, each method is compared and evaluated against the "total folate" results provided by routine radioassay and microbiological assay determinations. On the basis of the experimental data presented in this report, it is suggested that both methods have the capacity to serve as potential reference methods for the quantification of circulating 5MT in plasma or serum.     

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.     

Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH₄)₂ SO₄ fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate.K _0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 ΜM, 2.45 ΜM and 16 ΜM. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.     

Folylpolyglutamate synthase and gamma-glutamyl hydrolase regulate leucovorin-enhanced 5-fluorouracil anticancer activity

Although 5-fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer, the factors that determine the LV-mediated enhancement of the antitumor activity of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV. LV enhanced the anticancer activity of 5-FU and the level of reduced folate in human colon cancer cells. Small-interfering RNA (siRNA) transfected into DLD-1 cells to downregulate FPGS reduced the basal level of reduced folate, the folate level after LV treatment, and the enhancement of 5-fluoro-2′-deoxyuridine (FdUrd)-induced cytotoxicity elicited by LV. By contrast, the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results suggest that FPGS and GGH expression levels in tumors are determinants of the efficacy of LV in enhancing the antitumor activity of 5-FU.     

Affinity extraction combined with stable isotope dilution LC/MS for the determination of 5-methyltetrahydrofolate in human plasma

The predominant circulating folate monoglutamate in human plasma (>90%), and thus the most significant folate for accurately diagnosing folate deficiency, is 5-methyltetrahydrofolic acid (5 MT). Folate deficiency is typically indicated when circulating folate levels are < or = 3 ng/mL. The quantitative determination of plasma folates in general, and of 5 MT in particular, is complicated by their naturally low levels (pg/mL to ng/mL), their instability, and their tendency to interconvert. Highly specific and sensitive analytical methods are needed to accurately quantify endogenous 5 MT in human plasma. A method that utilizes the specific high-affinity binding sites of bovine folate binding protein (FBP) and the selectivity and sensitivity of selected ion monitoring mode isotope-dilution liquid chromatography/mass spectrometry (LC/MS) to quantify plasma 5 MT has been developed. The method is based on the solid-phase affinity extraction (SPAE) of 5 MT and its stable isotopically labeled analogue ([13C(5)]5 MT) from plasma (1 mL) using FBP immobilized to polymeric beads. The excess high-affinity binding sites on the affinity columns enable quantitative extraction of 5 MT from plasma under optimized sample pH conditions. Additionally, it is demonstrated that plasma proteins do not hinder the determination of 5 MT; therefore, protein precipitation is not required before the affinity extraction step. Detection and quantification of the extracted 5 MT is provided by positive-ion mode LC/MS in which the protonated molecular ions [M+H](+) of the analyte and the internal standard are monitored. The method shows linearity over three orders of magnitude (0.04-40 ng/mL) and has limits of detection and quantification of 0.04 and 0.4 ng/mL, respectively. Calibration curves obtained by spiking 5 MT into plasma exhibited good linearity between 0 and 25 ng/mL and both the plasma calibration standards and the plasma samples were stable for at least 48 h at room temperature. The recovery (average +/- % RSD) of 5 MT spiked into plasma from 5 to 25 ng/mL was 98.0% +/- 1.6% (n = 15). 5 MT levels determined by SPAE-LC/MS compared to "total folate" levels determined by radioassay and microbiological assay were discordant. Reasons for the discordancy are theorized, but it is clear that there exists an urgent need for clinical reference materials containing certified folate levels.     

The utilization of formate by human erythrocytes

The incorporation of radioactive formate into an acid-stable non-volatile form by human erythrocytes is dependent upon the addition of 5-amino-4-imidazolecarboxamide riboside. The formate-incorporating activity of human erythrocytes varies widely among normal individuals and the values obtained are characteristic of the erythrocytes obtained from these individuals. The variation is unrelated to the total folate levels of the erythrocytes as measured by the growth response of Lactobacillus casei but is roughly correlated with the quantity of folate forms in the erythrocytes which support the growth of Steptococcus faecalis. The activities of several enzymes involved in the metabolism of the folate coenzymes has also been measured in extracts of erythrocytes. Extracts from all the individuals contained 10-formyltetrahydrofolate synthase, 5-amino-4-imidazolecarboxamide ribotide transformylase, and 5,10-methylenetetrahydrofolate dehydrogenase. None of the extracts contained detectable quantities of either 5,10-methylenetetrahydrofolate reductase or 5-methyltetrahydrofolate-homocysteine methyltransferase. These data support the conclusion that 5-methyltetrahydrofolate is not in metabolic equilibrium with the other forms of folate in the erythrocyte and the uptake of formate by intact erythrocytes is a function of those forms of the folate coenzymes which can be converted to tetrahydrofolate.     

Assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring 5-methyltetrahydrofolate and tetrahydrofolate using high-performance liquid chromatography with fluorescence detection.

We developed a method for assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring their products of 5-methyltetrahydrofolate (5-CH(3)-H(4)folate) and tetrahydrofolate (H(4)folate) directly, using high-performance liquid chromatography with fluorescence detection. Folate derivatives and enzymes were stable in the assay process. No reagents in the assay mixture were found to disturb the separation and detection of both H(4)folate and 5-CH(3)-H(4)folate in our assay system. The detection limit of this method was less than 20 nM H(4)folate or 5-CH(3)-H(4)folate in the enzyme assay system. This analytical method, therefore, has a sensitivity high enough to obtain accurate parameters of Michaelis-Menten kinetics and for assays of crude extracts from various biological samples. In addition, the analytical procedure is very simple and economical; it may be a useful tool for studying methylenetetrahydrofolate reductase and methionine synthase activities.     

Evidence for the existence of covalent nucleotide-thymidylate synthase complexes, identification of site of attachment, and enhancement by folates

The formation of covalent binary complexes of thymidylate synthase and its nucleotide substrate dUMP, product dTMP, and inhibitor, 5-fluorodeoxyuridylate (FdUMP) was investigated using the trichloroacetic acid precipitation method. It was observed that, in addition to FdUMP, both dUMP and dTMP were capable of covalent interactions with the enzyme in the absence of added folates. The presence of folate, dihydrofolate, or tetrahydrofolate (H4folate) was found to produce substantial enhancements in the covalent binding of both FdUMP and dUMP to the enzyme with H4folate being the most effective agent. Further, covalent binary complexes of the enzyme with the three radiolabeled nucleotides were isolated by trichloroacetic acid precipitation and subjected to CNBr cleavage. The active-site CNBr peptide was isolated by reverse phase high performance liquid chromatography, and the first five N-terminal amino acid residues were sequenced by the dansyl-Edman procedure. Each active site peptide obtained from the covalent binary complexes as well as that from the covalent inhibitory ternary complex formed from enzyme, FdUMP, and 5,10-methylene-H4folate exhibited an identical sequence of Ala-Leu-Pro-Pro-(X)-, and the 5th amino acid was found to be associated with radiolabeled nucleotide ligand. Dansyl-Edman sequence analysis of the active site CNBr peptide, derived from enzyme which had been treated with iodoacetic acid, gave a sequence of Ala-Leu-Pro-Pro-CmCys (where CmCys is carboxymethylcysteine), thus confirming the fact that the fifth residue from the N terminus is Cys-198. In all the cases, the active site Cys-198 residue was found to be covalently linked to the nucleotides. These results provide unequivocal proof that the covalent binary complexes of enzyme with dUMP and dTMP predicted in the catalytic reaction mechanism actually exist.     

Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.     

Utilization of yeast polyglutamate folates in man.

Ingestion by healthy humans of small amounts of polyglutamate folates from yeast, equivalent to 300 mug of monoglutamate folate and containing 30 mug of "free folate," resulted in an appreciable elevation of the serum folate corresponding to 300 mug of synthetic pteroylmonoglutamate (PGA). Ingestion of higher amounts of polyglutamate folate did not result in higher serum folate elevations than did 300 mug. It is concluded that small amounts of polyglutamate folate from yeast are fully utilized, presumably by deconjugation in the gut prior to absorption. The relative ineffectiveness of larger doses of polyglutamate folates from yeast may be due to limiting conjugase activity in the gut, unfavorable conditions for its activity (such as unsuitable pH) or to an inhibitor of the enzyme present in impure preparations.     

Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.