As shown in Figure 10.4, 10-formyl-tetrahydrofolate and methylene-tetra-hydrofolate are donors of one-carbon fragments in a number of biosynthetic reactions, including the synthesis of purines, pyrimidines, porphyrins, and
the methylation of homocysteine to methionine. In most cases, the reaction is a simple transfer of the one-carbon group from substituted tetrahydrofolate onto the acceptor substrate.
Two reactions are of special interest: thymidylate synthetase (Section 10.3.3) and remethylation of homocysteine to methionine (Section 10.3.4). This latter reaction is central to the control of the metabolism of one-carbon compounds and folate.
10.3.3.1 Thymidylate Synthetase and Dihydrofolate Reductase Methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP; see Figure 10.8) is essential for the synthesis of DNA, although preformed TMP can be reutilized by salvage from the catabolism of DNA.
The methyl donor is methylene-tetrahydrofolate. The reaction involves formation of a methylene bridge between N-5 of the coenzyme and C-5 of dUMP, followed by transfer of hydrogen from the pyrazine ring of tetrahydrofolate, thus forming dihydrofolate that is released from the enzyme. Dihydrofolate is a product of this reaction, whereas in methylene-tetrahydrofolate reductase (Section 10.3.2.1), the dihydrofolate is a transient enzyme-bound intermediate and is reduced back to tetrahydrofolate during the reaction.
5-Fluorouracil is widely used in cancer chemotherapy. It is a precursor of 5-fluoro-dUMP, which is a mechanism-dependent inhibitor of thymidylate synthetase. It forms a stable methylene-bridged complex with methylene-tetrahydrofolate on the enzyme catalytic site that cannot undergo reductive cleavage.
10.3.3.2 Dihydrofolate Reductase Inhibitors Under normal conditions, the dihydrofolate formed by thymidylate synthetase is rapidly reduced to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthetase and di-hydrofolate reductase are especially active in tissues with a high rate of cell division, and thus a high rate of DNA replication and a high requirement for thymidylate. As cells enter the S-phase of the cell cycle, there is a 7fold increase in the rate of transcription of the dihydrofolate reductase gene (Farnham and Schimke, 1985). Because of this role in actively dividing tissue, inhibitors of dihydrofolate reductase have been exploited as anticancer drugs. The most successful of these is methotrexate, the 4-amino analog of 10-methyl-tetrahydrofolate (see Figure 10.1).
Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dihydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as 5-formyl-tetrahydrofolate, leucovorin) to replete the normal tissues and avoid induction of folate deficiency- so-called leucovorin rescue. As well as depleting tissue pools oftetrahydrofolate, methotrexate leads to the accumulation of relatively large amounts of 10-formyl-dihydrofolate, which is apotentinhibitor of both thymidylate synthetase and glycinamide ribotide transformylase, an intermediate step in purine nucleotide synthesis. It is likely that this, rather than simple depletion of tetrahydrofolate, is the basis of the cytotoxic action of methotrexate (Barametal., 1988).
Methotrexate is also a substrate for conjugation with glutamate (Section 10.2.2.1) and a variety of methotrexate polyglutamates, which are potent inhibitors of dihydrofolate reductase are formed and retained in the cells. Susceptible tumor cells show greater conjugation, and greater accumulation, of methotrexate than bone marrow cells or the gastrointestinal mucosa, thus providing some degree of tumor specificity for the drug. Methotrexate polyglutamates inhibit the reactivation of dihydrofolate reductase by 5-formyl-
tetrahydrofolate so that leucovorin rescue has less effect on (tumor) cells that accumulate and conjugate methotrexate (Goldman and Matherly, 1987).
The antibacterial agent trimethoprim also acts as an inhibitor of dihydro-folate reductase. It binds to the bacterial enzyme with much higher affinity than the mammalian enzyme. It provides a considerable degree of selectivity, permitting use of doses low enough to have little effect on the host's folate metabolism. A number of trimethoprim-resistant strains of bacteria have been isolated; at least three different plasmid-associated dihydrofolate reductases have been identified, including the following:
1. low K for trimethoprim and thus insensitive to inhibition;
2. sensitive to trimethoprim inhibition, but with a lower Km for dihydrofo-late, which therefore competes with the drug more effectively;
3. sensitive to trimethoprim and with a high Km for dihydrofolate, but induced by trimethoprim, thus increasing the amount of enzyme available.
10.3.3.3 The dUMP Suppression Test Rapidly dividing cells can either use preformed TMP or can synthesize it de novo from dUMP. Isolated bone marrow cells or stimulated lymphocytes incubated with [3H]TMP will incorporate label into DNA. In the presence of adequate amounts of methylene-tetrahydrofolate, the addition of dUMP as a substrate for thymidylate synthetase reduces the incorporation of [3 H]TMP as a result of dilution of the pool of labeled material by newly synthesized TMP.
The extent to which dUMP suppresses the incorporation of [3H]TMP into DNA thus reflects folate nutritional status (Section 10.10.5).
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