Thymidylate synthetase activities

Methotrexate is a folic acid analogue. Its mechanism of action is based on the inhibition of dihydrofolate reductase. Inhibition of dihydrofolate reductase leads to depletion of the tetrahydrofolate cofactors that are required for the synthesis of purines and thymidylate (see Fig. 2). Enzymes that are required for purine and thymidylate synthesis are also directly inhibited by the polyglutamates of methotrexate which accumulate with dihydrofolate reductase inhibition. The mechanisms that can cause resistance include decreased transport of methotrexate into the tumor cells, a decreased affinity of the antifolate for dihydrofolate reductase, increased concentrations of intracellular dihydrofolate reductase and decreased thymidylate synthetase activity. 

Approximately 6% of a topically applied dose is absorbed—an amount insufficient to produce adverse systemic effects. Most of the absorbed drug is metabolized and excreted as carbon dioxide, urea, and ct-fluoro-B-alanine. A small percentage is eliminated unchanged in the urine. Fluorouracil inhibits thymidylate synthetase activity, interfering with the synthesis of DNA and, to a lesser extent, RNA. These effects are most marked in atypical, rapidly proliferating cells. 

Experimental animals that have been exposed to ititrous oxide to deplete vitamin B12 show an increase in the proportion of liver folate present as methyl-tetrahydrofolate (85% rather than the normal 45%), largely at the expense of unsubstituted tetrahydrofolate and increased urinary loss of methyl-tetrahydrofolate (Horne et al., 1989). Tissue retention of folate is impaired because methyl-tetrahydrofolate is a poor substrate for polyglutamyl-folate synthetase, compared with unsubstituted tetrahydrofolate (Section 10.2.2.1). As a result of this, vitamin B12 deficiency is frequently accompanied by biochemical evidence of functional folate deficiency, including impaired metabolism of histidine (excretion of formiminoglutamate Section 10.3.1.2) and impaired thymidylate synthetase activity (as shown by abnormally low dUMP suppression Section 10.3.3.3), although plasma concentrations of methyl-tetrahydrofolate are normal or elevated. 

Hall, S. J., Sims, P. F., and Hyde, J. E. (1991). Functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the human malaria parasite Plasmodium falciparum in Escherichia coli. Mol. Biochem. Parasitol. 45,317-330. 

Inasmuch as TTP, one of the four triphosphate nucleotides needed for DNA synthesis, is formed through the methylation of UMP to TMP which is then further phosphorylated, one may expect that a reduction in methyl transfer would reduce the levels of TTP and thereby cause interference with DNA synthesis and maturation of the red cell. Yet, the pools of TTP in lymphocytes were normal in untreated patients deficient in vitamin Bi2- In contrast, in patients treated with methotrexate a marked drop in the TTP pool is found [166]. A drop in thymidylate synthetase activity in phytohemagglutinin stimulated lymphocytes of patients with pernicious anemia has been described. 

Because of the rapid synthesis of DNA during differentiation, lymphoid cells would be expected to be particularly sensitive to abnormalities in the regulation of the nucleoside pools that serve as precursors for DNA synthesis. The synthesis of thymidine triphosphate is of particular interest because of its specificity for DNA synthesis. The rate limiting step in the de novo pathway to dUMP involves conversion of dUPM to a dTMP by the folate requiring enzyme thymidylate synthetase, vitro incorporation of 3H-thymidine and 3n-deoxyuridine is reduced in bone marrow cultures from folic acid deficient rats (Sakamato and Takaku, 1973). Thymidylate synthetase activity was elevated when assayed in the presence of adequate cofactor (5,10-methylene THF). A salvage pathway also exists for the direct phosphorylation of thymidine by thymidine kinase. This enzyme can exist in two forms (Ellims et al., ... 

In folate or vitamin B12 deficiency one would expect to see changes in the levels of activity of the folate (and indirectly 12) dependent enzyme thymidylate synthetase, Haurani (1973) reported low or non-existant thymidylate synthetase activity in PHA stimulated lymphocytes from vitamin Bj 2 not folate) deficient patients. 

Conrad, A. H. and Ruddle, F. H. (1972) Regulation of thymidylate synthetase activity in cultured mammalian cells, J. Cell, Sci., 10 471. 

Sakamato, S. and Takaku, F. (1973) Thymidylate synthetase activity in bone marrow cells of folic acid deficient rats, Acta. Hematol. Jap, 36 556. 

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