Tetrahydrofolate from dihydrofolate reductase

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

Both the sulfonamides and trimethoprim interfere with bacterial folate metabolism. For purine synthesis tetrahydrofolate is required. It is also a cofactor for the methylation of various amino acids. The formation of dihydrofolate from para-aminobenzoic acid (PABA) is catalyzed by dihydropteroate synthetase. Dihydrofolate is further reduced to tetrahydrofolate by dihydrofolate reductase. Micro organisms require extracellular PABA to form folic acid. Sulfonamides are analogues of PABA. They can enter into the synthesis of folic acid and take the place of PABA. They then competitively inhibit dihydrofolate synthetase resulting in an accumulation of PABA and deficient tetrahydrofolate formation. On the other hand trimethoprim inhibits dihydrofolate... 

Conversion of dUMP to dTMP is catalyzed by thy-midylate synthase. A one-carbon unit at the hydroxymethyl (—CH2OH) oxidation level (see Fig. 18-17) is transferred from Af5,Af10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group (Fig. 22-44). The reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate, which is unusual in tetrahydrofolate-requiring reactions. (The mechanism of this reaction is shown in Fig. 22-50.) The dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase—a regeneration that is essential for the many processes that require tetrahydrofolate. In plants and at least one protist, thymidylate synthase and dihy-drofolate reductase reside on a single bifunctional protein. 

Like the biguanides it is a potent inhibitor of dihydrofolate reductase of the plasmodium (mammalian enzyme is about 200 times less sensitive). Thus it blocks the synthesis of tetrahydrofolic from dihydrofolic acid and this is essential for the synthesis of purines and pyrimidines and hence DNA. 

Cells that are synthesizing DNA mnst also be able to synthesize deoxy thymidine triphosphate (dTTP). The key step in the synthesis is the conversion of dUMP to dTMP via thymidylate synthetase. The reaction reqnires a sonrce of N, -methylene tetrahydrofolate to provide the methyl gronp. In this reaction the tetrahydrofolate is oxidized to dihydrofolate. Dihydrofolate is rednced to tetrahydrofolate via dihydrofolate reductase so more methylene If, A °-tetrahydrofolate is made from serine in a reaction that is catalyzed by serine hydroxymethyltransferase. These three reactions, which are essential for the formation of dTMP, are shown in Fig. 14-20. 

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.)...

In a collaboration between the Abelson and Hecht labs [56b], a series of noncoded amino acids were introduced into dihydrofolate reductase (DHFR) to probe substrate binding and the requirement of an aspartic acid residue for catalytic competence. When aspartic acid analogs mono- or disubstituted at the )0-carbon were substituted for the active site aspartic acid residue, the mutant DHFRs were still able to catalyze the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate at 74 - 86 % of the wild-type rate. While hydride transfer from NADPH is not the rate-limiting step for the wild-type enzyme at physiological pH, a kinetic isotope experiment with NADPD indicated that hydride transfer had likely become the rate-limiting step for the mutant containing the )0,)0-dimethylaspartic acid. 

Inhibition of nucleobase synthesis (2). Tetrahydrofolic acid (THF) is required for the synthesis of both purine bases and thymidine. Formation of THF from folic acid involves dihydrofolate reductase (p. 272). The folate analogues aminopterin and methotrexate (ame-thopterin) inhibit enzyme activity as false substrates. As cellular stores of THF are depleted, synthesis of DNA and RNA building blocks ceases. The effect of these antimetabolites can be reversed Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of iicense. 

It acts by inhibiting dihydrofolate reductase. It inhibits conversion of dihydrofolic acid to tetrahydrofolic which is essential for purine synthesis and amino acid interconversions. It primarily affects DNA synthesis but also RNA and protein synthesis. It has cell cycle specific action and kills cells in S phase. It is readily absorbed from gastrointestinal tract but larger doses are absorbed incompletely, little drug is metabolised and it is excreted largely unchanged in urine. 

The active form of folic acid, tetrahydrofolic acid (THF), is produced from folate by dihydrofolate reductase in a two-step reaction requiring two moles of NADPH. The carbon unit carried by THF is bound to nitrogen N5 or N10, or to both N5 and N10. THF allows one-carbon compounds to be recognized and manipulated by biosynthetic enzymes. Figure 20.11 shows the structures of the various members of the THF family, and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate. 

Figure 15-19 Drawings of the active site of E. coli dihydrofolate reductase showing the hound ligands NADP+ and tetrahydrofolate. 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.381 Courtesy of Joseph Kraut. Molscript drawings (Kraulis, 1991).

Methotrexate acts by inhibition of dihydrofolate reductase, the enzyme requisite for the reduction of dihydrofolic acid (3) to 5,6,7,8-tetrahydrofolic acid (4). In turn, (4) is a precursor to a series of enzyme cofactors (5-7) essential for the transfer of one carbon unit necessary for the biosynthesis of purines and pyrimidines and hence, ultimately, DNA. As an inhibitor of dihydrofolate reductase, methotrexate kills cells during the S phase of the cell cycle, when the cells are in the log phase of growth. Unfortunately, this cytotoxicity is non-selective, and rapidly proliferating normal cells, e.g., gastrointestinal epithelium cells and bone marrow, are dramatically affected as well. In addition, recent use of high dose methotrexate therapy with leucovorin rescue has led to additional clinical problems arising from a dose-related nephrotoxic metabolite, 7-hydroxy methotrexate (8). Finally, the very polar nature of methotrexate renders it virtually impenetrable to the blood-brain barrier, which can necessitate direct intrathecal injection in order to achieve therapeutic doses for the treatment of CNS tumours. 

Mammals must obtain their tetrahydrofolate requirements from their diet, but microorganisms are able to synthesize this material. This offers scope for selective action and led to the use of sulphanilamide and other antibacterial sulpha drugs, compounds which competitively inhibit dihydropteroate synthase, the biosynthetic enzyme incorporating p-aminobenzoic acid into the structure. These sulpha drugs thus act as antimetabolites of p-aminobenzoate. Specific dihydrofolate reductase inhibitors have also become especially useful as antibacterials,... 

The donation of the methyl group from N5,N10-methylene tetrahydrofolate leads to the oxidation of the cofactor to dihydrofolate. This points to the importance of dihydrofolate reductase (DHFR) in the functioning of thymidylate synthase. Thus, synthesis of TMP requires a supply of both methyl groups—for example, from serine— and reducing equivalents. 

Aminopterin and amethopterin are 4-amino analogues of folic acid (Fig. 11.5) and as such are potent inhibitors of the enzyme dihydrofolate reductase (EC 1.5.1.3) (Blakley, 1969). This enzyme catalyses the reduction of folic acid and dihydrofolic acid to tetrahy-drofolic acid which is the level of reduction of the active coenzyme involved in many different aspects of single carbon transfer. As is clear from Fig. 11.6, tetrahydrofolate is involved in the metabolism of (a) the amino acids glycine and methionine (b) the carbon atoms at positions 2 and 8 of the purine ring (c) the methyl group of thymidine and (d) indirectly in the synthesis of choline and histidine. 

S)-Tetrahydrofolate was used as starting material for the synthesis of L-leu-covorin. Applying an NADPH-dependent dihydrofolate reductase in combination with GDH from Gluconobacter scleroides KY3613 for recycling of the cofactor, (6S)-tetrahydrofolate was synthesized from dihydrofolate [48]. 

Tetrahydrofolic acid (THF) is a coenzyme in the synthesis of purine bases and thymidine. These are constituents of DNA and RNA and are required for cell growth and replication. Lack of THF leads to inhibition of cell proliferation. Formation of THF from dihydrofolate (DHF) is catalyzed by the enzyme dihydrofolate reductase. DHF is made from folic acid, a vitamin that cannot be synthesized in the body but must be taken up from exogenous sources. Most bacteria do not have a requirement for folate, because they are capable of synthesizing it-more precisely DHF-ffom precursors. Selective interference with bacterial biosynthesis of THF can be achieved with sulfonamides and trimethoprim. 

Tetrahydrofolate is regenerated from the dihydrofolate that is produced in the synthesis of thymidylate. This regeneration is accomplished by dihydrofolate reductase with the use of NADPH as the reductant. 

FIGURE 17.26. Binding of (a) methotrexate to dihydrofolate reductase and (b) a comparison with a model of tetrahydrofolate bound in the same orientation, (c) The binding mode observed for NADPH and methotrexate in the crystalline state. It is seen in (c) that when tetrahydrofolate is bound in the same way as methotrexate, the hydrogen atom cannot be transferred, (d) Therefore the tetrahydrofolate must be bound in a different manner from methotrexate for a reaction to occur. The two orientations of the two ligands are related by a rotation. This has been verified by an X-ray structure determination (Ref. 122). Thus tetrahydrofolate and methotrexate do not bind in the same manner to the enzyme. 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

Fig. 12.7 Pathways of folate metabolism and use in microbial cells (upper) and mammalian cells (lower). Bacterial and protozoal cells must synthesize dihydrofolic acid (DHF) from p-aminobenzoic acid (PABA). DHF is converted to tetrahydrofolic acid (THF) by the enzyme dihydrofolate reductase (DHFR). THF supplies single carbon units for various pathways including DNA, RNA and methionine synthesis. Mammalian cells do not make DHF, it is supplied from the diet, conversion to THF occurs via a DHFR enzyme as in microbial cells.

Methotrexate inhibits DNA synthesis by decreasing avail-ability of pyrimidine nucleotides. Methotrexate competitively inhibits the enzyme dihydrofolate reductase, thus decreasing the concentrations of the tetrahydrofolate essential to the methylation of the pyrimidine nucleotides and consequently the rate of pyrimidine nucleotide synthesis. Leucovorin, a folate analog, is used to rescue host cells from methotrexate inhibition as a synthetic substrate for dihydrofolate reductase, leucovorin administration allows resumption of tetrahydrofolate-dependent synthesis of pyrimidines and reinitiation of DNA synthesis. Methotrexate is a nonspecific cytotoxin, and prolongation of blood levels appropriate to killing tumor cells may lead to severe, unwanted cytotoxic effects such as myelosuppression, gastrointestinal mucositis, and hepatic cirrhosis. 

Tetrahydrofolate (Fig. le) and derivatives (Fig. If, g) are the biologically active forms of folate. This cofactor is involved in many, distinct enzymatic reactions, ranging from the amino acid metabolism, such as serine hydroxymethyltransferase (SHMT) (Fig. 5a), to nucleotide biosynthesis, such as thymidylate synthase (TS) (Fig. 5b) and dihydrofolate reductase DHFR (Fig. 5c). These enzymes are targets for anticancer drugs because they participate in the formation of thymidylate, the only nucleotide that cannot be obtained via the salvage reactions (30). Whereas the search for inhibitors of SHTM has only recently... 

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