Tetrahydro folate

Hill et al. (141) hrst observed that light-stable yellow alkylcorrinoids could be synthesized. When methylcobalamin was treated with picrate, a yellow corrinoid was isolated which was shown to be stable to light, but unstable to cyanide. Similarly, Taylor and Weissbach (67) demonstrated that the methylcorrinoid-enzyme complex of N5-methyl-tetrahydro-folate-homocysteine transmethylase was stable to light, but isolation of... 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

Figure 20.12 (a) Details of reaction catalysed by thymidylate synthase. Methylene FH4 represents N N methylene tetrahydro-folate (see Figure 15.2). (b) Reactions in the pathways in which either CDP or UDP gives rise to deoxythymidine monophosphate. Note that two processes can be involved in synthesis of deoxyuridine monophosphate. It is not known if one process dominates, but in (c) it is assumed that the pathway from CDP dominates formation of dTTP. (c) k summary of the reactions required for synthesis of deoxyribonucleotides required for DNA replication. 

Serine as a Cl donor. For many organisms, from E. coli to higher animals, serine is a major precursor of C-l units.395-397 The (3-carbon of serine is removed as formaldehyde through direct transfer to tetrahydro-folate with formation of methylene-THF and glycine (Eq. 14-30, Fig. 15-18, step c). This is a stereospecific transfer in which the pro-S hydrogen on C-3 of serine enters the pro-S position also in methylene-THF.398 The glycine formed in this reaction can, in turn, yield... 

Thymidylate synthase requires methylene tetrahydro-folate as a reductant and the reduction of dihydrofolate is also an important part of the process. In protozoa dihydrofolate reductase and thymidylate synthase occur as a singlechain bifunctional enzyme.f As has been pointed out in the main text, such folic acid analogs as methotrexate are among the most useful anticancer drugs. By inhibiting dihydrofolate reductase they deprive thymidylate synthase of an essential substrate. 

In addition to the larger families of preparatively useful aldolases, some less common aldolases have been evaluated lately for preparative use. A range of mechanistically distinct enzymes, which are actually categorized as transferases but which also catalyze aldol-related additions through the aid of cofactors such as pyridoxal 5-phosphate (PLP), thiamine pyrophosphate (TPP), tetrahydro-folate (THF), or coenzyme A (CoA), are becoming more frequently applied in organic synthesis. Because of their emerging importance and/or commercial availability, a selection of these enzymes and examples of their synthetic utility will be included in further separate chapters. 

The velocity of a reaction is therefore proportional to the total amount of enzyme (Et) in the previous reaction. If the activity of dihydrofolate reductase were to be inhibited tenfold by methotrexate, increasing Et tenfold can restore the velocity (amount of tetrahydro-folate made per time). 

In the folate coenzymes, the pteridine ring is fully reduced to tetrahydro-folate, although the oxidized form, dihydrofolate, is an important metabolic intermediate. In the reactions of thymidylate synthetase (Section 10.3.3) and methylene tetrahydrofolate reductase (Section 10.3.2.1), the pteridine ring has a redox role in the reaction. The folate coenzymes are conjugated with up to six additional glutamate residues, finked by y-glutamyl peptide bonds. 

Most dietary folate is reduced and methylated to methyl-tetrahydro-folate in the intestinal mucosa (Section 10.2.1). Intestinal mucosal ceUs have a rapid turnover, typicaUy 48 hours from proliferation in the crypt to shedding at the tip of the vUlus. This means that an unstable variant of the enzyme, which loses activity over a shorter time than the normal enzyme, is probably irrelevant in ceUs that have such a rapid turnover. A high intake of folate would therefore result in a relatively high rate of supply of methyl-tetrahydrofolate to cells, arising from newly absorbed folate, so that impaired turnover of folate within cells would be less important. 

The possible relationship of the methane fermentation with the more conventional examples of one-carbon metabolism as catalyzed by folate and vitamin B12 cofactors has been long apparent. 5-Methyl tetrahydro-folate, 5,10-methylene tetrahydrofolate, and methyl vitamin B12 are converted to methane by cell-free extracts of M. barkeri 32) and M. omeli-anskii (33). The involvement of vitamin B12 is further implicated by its high cellular level in methane bacteria and by the isolation of B12-containing proteins in extracts of M. barkeri 30) which stimulate methane evolution from methyl vitamin B12. The components and pathways that can be demonstrated in cell-free M. barkeri extracts 32) are listed below. 

Figure 15-19 Drawings of the active site of E. coU dihydrofolate reductase showing the bound ligands NADP+ and tetrahydro-folate. Several key amino acid side chains are shown in the stereoscopic views on the right. The complete ribbon structures are on the left. (A) Closed form. (B) Open form into which substrates can enter and products can escape. From Sawaya and Kraut. Courtesy of Joseph Kraut. Molscript drawings (Kraulis, 1991).

Methotexate is an antineoplastic folic add analogue that blocks the conversion of dihydrofolate (FHj) to tetrahydro-folate (FH4) by binding to dihydrofolate reductase (DHFR) enzyme. Folate is essential for the normal synthesis of purines and pyrimidines, and therefore DNA and RNA. In order for folate to function as a cofactor, it must be reduced to FH by DHFR. Methotrexate binds to DHFR, prevents the conversion of FH2 to FH4, and, consequently, inhibits purine and pyrimidine synthesis. The antimetabolites are considered cell cycle specific, with most activity for cells in the S (synthesis) phase. With high-dose methotrexate, leucovorin rescue is often used to prevent severe toxicity to normal body tissues. Leucovorin (folinic acid) is a reduced form of folate (similar to FH ) that does not require the use of DHFR. Leucovorin is transported into healthy cells and is utilized for DNA and RNA synthesis. Tumor cells tend to have impaired transport mechanisms and usually cannot use leucovorin. Leucovorin is usually started within 24 to 36 hours of high-dose methotrexate administration and continues until methotrexate serum levels are below nontoxic levels (0.1 to 0.05 mol/L). 

The dihydrofolate reductase inhibitor, methotrexate (Fig. 8.47), was developed as an anticancer drug, whose inhibition of formation of folic acid coenzymes would block purine synthesis. In other words, it was designed to induce a folic acid deficiency. Notice in Figs. 8.50 and 8.51 that formation of dTMP, FGAR, and AICAR also causes the oxidation of tetra-hydrofolate to dihydrofolate. The latter must be reduced by dihydrofolate reductase to tet-rahydrofolate before active coenzyme can form again. Thus, not only does methotrexate inhibit the initial formation of the tetrahydro-folate moiety, it blocks regeneration of the coenzyme form. 

G14. Grignani, F., Martelli, M. F., Tonato, M., and Finzi, A. F., Folate dependent enzymes in human epidermis Tetrahydro-folate dependent enzymes and relationship between dehydrofolate reductase and anethopterin. Arch. Dermatol. 96, 577-585 (1967). 

In the synthesis of methionine, cob(I)alamin is methylated by methyl tetrahydro-folate [MeHjTHF (equation (a)], followed by the transfer of CHj " from methylcoba-lamin to homocysteine . ... 

Pyrimethamine is a folic-acid antagonist its therapeutic action is based on differential requirements between host and parasite for nucleic acid precursors involved in growth as it selectively inhibits plasmodial dihydrofolate reductase. Pyrimethamine inhibits the enzyme dihydrofolate reductase that catalyzes the reduction of dihydrofolate to tetrahydro-folate. This activity is highly selective against plasmodia and Toxoplasma gondii. It does not destroy gametocytes but arrests sporogony in the mosquito. Pyrimethamine possesses a blood schizonticidal, and some tissue schizonticidal activity may be slower than that of 4-amino-quinoline compounds. 

A particularly elegant method has recently been devised for the stereoselective synthesis of (/ -methyl)- and (S-methyl)-yV -[merAy/- H, H, H]tetrahydro-folate (N -CHa-THF) for examining the stereochemistry of THF-dependent enzymes (344). For example, synthesis of the former substrate first involves reduction of Af, A -methenyl-THF with NaB Ha, demonstrated to be approximately 80% stereoselective on the basis of proton nuclear Overhauser effect measurements on the product (346) [Eq. (63)] ... 

N-Methylation yields the monomethyl derivative sarcosine and also dimethylglycine, compounds that may function as osmoprotectants (Box 20-C). Many bacteria produce sarcosine oxidase, a fla-voprotein that oxidizes its substrate back to glycine and formaldehyde, which can react with tetrahydro-folate. The formation of porphobilinogen and the various pyrrole pigments derived from it and the synthesis of the purine ring (Chapter 25) represent two other major routes for glycine metabolism. 

Methotrexate and aminopterin, a similar compound, are analogs of dihydro folate (DHF) and inhibitors of dihydrofolate reductase, an enzyme that converts DHF to tetrahydro-folate (THF). The thymidylate synthase reaction converts N, N °-methylenetetrahydro-folate to DHF in the process of methylating dUMP to form dTMP In the presence of one of the inhibitors, this reaction functions as a sink that reduces the THF level of the cell by converting THF to DHF. Since THF derivatives are substrates in two reactions of purine metabolism and one of pyrimidine metabolism, both pathways are affected by the inhibitor. 

The tetrahydropterin-dithioiene iigand generated considerabie excitement and speculation. It was unique in biochemistry, and is unusual in chemistry. While dithiolenes were well-known ligands for molybdenum and other metals, this was the first time a dithioiene was proposed to play a role in biochemistry. On the other hand, tetrahydropterins were already known molecules in biochemical processes, such as the tetrahydrobiop-terin cofactor used by aromatic amino acid hydroxylases and tetrahydro-folate in Cl transfer in methionine synthesis (Figure 2.3). Certainly, this was the first time a pterin was found to be in combination with a dithioiene an where in chemistry. 

Phosphoribosyl-AT-formyl-glycineamide tetrahydro-folate 5,10-formyl transferase Phosphoribosyl glycineamide formyltransferase... 

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