Methylenetetrahydrofolic acid

Fig. 1. Chemical structure of cofactors (a) NAD(P)+ and (b) NADH(P), with R = H is NAD+ or NADH, and R = P is NADP+ or NADPH, (c) Flavin mononucleotide (FMN), (d) Flavin adenine dinucleotide (FAD), (e)Tetrahydrofolate, (f) 10-formyltetrahydro-folic acid, (g) 5-10 methylenetetrahydrofolic acid,...

The (3R)- and (3S)-[3- Hi,3- Hi]serines 60, Hj, = H, H = H, and 60, H = H, Hj = H, respectively, were also used with the coupled enzymes serine hydroxymethyltransferase and methylenetetrahydrofolate reductase (EC 1.1.1.171) (100). Enzymic reduction of the labeled samples of the intermediate 5,10-methylenetetrahydrofolic acid 56b gave samples of 5-methyltetrahydrofolic acid 91 (Scheme 26). These were degraded to acetate by a sequence that involved one inversion of configuration, and assay of the acetates showed that the overall stereochemistry of the reduction was as in Scheme 26 (100). 

L-Serine is the most important source of one-carbon units. In the presence of THF it is degraded by serine hydroxymethyltransferase to glycine and 5,10-methylenetetrahydrofolic acid ... 

Glycine can either be converted to serine by addition of a methy lene group from Ns,N10-methylenetetrahydrofolic acid (see Figure 20.6A), or oxidized to C02and NH4+. 

Reaction of the tetrahydroquinoxalines 44 with triethyl orthoformate gave the bright-yellow formamidinium salts 45. Reduction of 45 (R = COaEt) with sodium borohydride in THF gave the colorless derivative 46 The latter compounds were also prepared from 44 by reaction with formaldehyde. The use of 5-formyluracil gave the substituted compound 47. ° These compounds were of interest as model systems for N, N °-methylenetetrahydrofolic acid (48). During these investigations the hydrolysis of the formamidinium salts 45 as a function of pH was... 

Biosynthesis of thymine nucleotides. This is shown in Fig. 2. Since thymine is a constituent of DNA, the corresponding nucleotides contain 2-deoxyribose. Thymidylic acid (TMP) is therefore more correctly dTMP (deoxythymidine 5 -monophosphate). The reaction sequence is CMP - CDP - dCDP -v dCMP ->dUMP- TMP (dTMP)- TDP (dTDP)- TTP (dITP). Methylation of dUMP to TMP is catalysed by th idylate synthase (EC 2.1.1.45). The cofiictor, AP,A/ -methylenetetrahydrofolic acid, transfers the active Cl unit to C5 of dUMP, and it also functions as a reducing agent in the formation of the methyl group from the active Cl unit. 

Folic acid (Fig. 6) is the precursor of a number of coenzymes vital for the synthesis of many important molecules. These derivatives of folic acid, referred to collectively as active formate and active formaldehyde , are responsible for the donation of one carbon fragments in the enzymatic synthesis of a number of essential molecules. In the formation of methionine from homocysteine, the folic acid coenzyme donates the S-methyl group, and in the conversion of glycine to serine it is necessary for the formation of the hydroxymethyl group. Folic add is converted into its active coenzyme forms by an initial two step reduction to tetra-hydrofolic add (Fig. 6) by means of two enzymes, folic reductase and dihydrofolic reductase. Conversion of tetrahydrofolic acid (THF) to an active coenzyme folinic acid subsequently occurs by ad tion of an Ns formyl group (Fig. 6). The formation of similar compounds such as an Nio formyl derivative, or the bridged Ns,Nio-methylenetetrahydrofolic acid, also leads to active coenzymes. 

TK), 5-FU is activated to 5-fluorodeoxyuridine monophosphate (5-FdUMP). Potent inhibition of thymidylate synthase (TS) by 5-FdUMP is considered critical for 5-FU cytotoxicity. TS catalyzes the rate-limiting step of DNA synthesis, such as the conversion of dUMP into dTMP. Optimal TS function requires the formation of a covalent ternary complex consisting of TS, the folate cofactor 5,10-methylenetetra-hydrofolate (CH2THF), and 5-FdUMP. Inadequate cellular levels of 5,10-methyle-netetrahydrofolate reduce the stability of the ternary complex and consequently the inhibition of TS by 5-FdUMP. For this reason, 5-FU is administered in association with folinic acid, a precursor of 5,10-methylenetetrahydrofolate [40]. 

In living systems, folinic acid can be synthesized ultimately from folic acid by reduction to tetrahydrofolic acid followed by addition of a 1-carbon fragment to the molecule (N5.N1°-methylenetetrahydrofolate, V). After a 2-step oxidation, the formyl group resides either at the N5 or N10 position or as an equilibrium mixture. The essential reactions are summarized below 32... 

Recently, the enzymatic formation of folinic acid has been utilized to synthesize radioactively labeled products.34 The preparation of 5-formyl tetrahydrofolate, 9,3, 5 -3H and 5-formyl-14C-tetrahydrofolate starts with tritiated folic acid, which is reduced to dihydrofolate, incubated in the presence of formaldehyde, dihydrofolate reductase, and NADPH, and finally incubated with 5,10-methylenetetrahydrofolate dihydrogenase. The product,... 

Formaldehyde is a toxic substance that reacts spontaneously with amino groups of proteins and nucleic acids, hydroxymethylating them and forming methylene-bridge crosslinks between them. Free formaldehyde therefore wreaks havoc in living cells and could not serve as a useful hydroxymethylating agent. In the form of A5,A10-methylenetetrahydrofolate, however, its chemical reactivity is attenuated but retained in a potentially available form where needed for specific enzymatic action. Formate, how-... 

N5,jV10-Methylenetetrahydrofolate (methylene-THF) is one of the folic acid coenzymes (Sec. 15.7). Note that this reaction is readily reversible, and in fact, net flux is usually in the direction of glycine synthesis. Thus this amino acid can arise from glucose, and it does so via serine. 

The enzyme assay contained in a total volume of 250 /xL 50 m M Tris-HCl buffer (pH 7.8), 50 mM 2-mercaptoethanol, 5 mil/ formaldehyde, and 1 mM methylenetetrahydrofolate. The reaction was initiated by adding 25 /xL of 1 mM deoxyuridylate. After 30 minutes at 37°C, the reaction was stopped by addition of 250 /xL of ice-cold 1 M perchloric acid. After 30 minutes in an ice bath, the reaction mixture was centrifuged and the resulting supernate was neutralized with 0.1 volume of 10 M KOH containing 1 M KH2PO4. The supernate obtained by centrifugation was injected onto the HPLC column. Formation of deoxythymidylate was linear with time up to 30 minutes, and with protein in the range of 80 to 720 /xg. 

There is at present no unifying concept which would explain the presence of optically active TIQs, TIQ-1-carboxylic acids, and noralkaloids in mammalian systems. The conclusion reached by several investigators that methylenetetrahydrofolate is responsible for the N-methylation of phenethylamines and indolylethylamines (221,222) makes it likely that the one-carbon unit present at C-1 in the noralkaloids is derived from formaldehyde, formed via nonenzymatic disassociation of methylene-tetrahydrofolates (223,224). 

Methylenetetrahydrofolate reductase (MTHFR) is encoded by the MTHFR gene located on chromosome lp36.22. It is the rate-limiting enzyme in the folic acid metabolic cycle that is critical for the... 

Thymidylate synthetase is an important enzyme, which is responsible for the reductive methylation of deoxyuridylic acid (dUMP, 7) to deoxythymidylic acid (dTMP, 8). The methylation of the uracil moiety (present in RNA) to 5-methyl uracil (thymine, present in DNA) requires participation of a folic acid coenzyme, N, methylenetetrahydrofolate as a methyl donor. The functioning of thymidylate synthetase is coupled with the activity of DHF reductase. That is why this biochemical target is usually referred to as thymidylate synthetase/ DHF reductase. 

For the synthesis of alkaloid D,L-pyridindolol 79, a diastereomeric mixture of the precursor ester 80 (79JOC535) has been conveniently obtained by the acid-catalyzed reaction of the 5,10-methylenetetrahydrofolate model imidazolidine 9 with methyl tryptophanate (83T3987). 

One of the most interesting diaminomethanes that has been investigated is a model for the coenzyme tetrahydrofolate (3), a biological one-carbon transfer agent. The mechanism of its conversion into the imidazolidine derivative /V, yV °-methylenetetrahydrofolate (4) has been studied. The pH rate profile apparently results from a change in the rate-determining step with changing acidity. Secondary amines such as morpholine and imidazole catalyze the reaction between tetrahydrofolic acid and formaldehyde via a... 

MECHANISM FIGURE 22-50 Conversion of dUMP to dTMP and its inhibition by FdUMP. The top row is the normal reaction mechanism of thymidylate synthase. The nucleophilic sulfhydryl group contributed by the enzyme in step (T) and the ring atoms of dUMP taking part in the reaction are shown in red B denotes an amino acid side chain that acts as a base to abstract a proton in step . The hydrogens derived from the methylene group of N, N -methylenetetrahydrofolate are shaded in gray. A novel feature of this reaction mechanism is a... 

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