Thymidylate synthetase 5,10-methylene tetrahydrofolate

A second relevant enzyme to 5-FU metabolism is thymidylate synthetase (TYMS). If this enzyme is complexed with 5-FU metabolites along with 5,10-methylene-tetrahydrofolate, it cannot maintain a thymidine-5 -monophosphate pool required for DNA replication and repair. A tandem repeat polymorphism in the 5 -promoter region of the TYMS gene can increase enzyme expression (85,86). Tumors carrying the repeats have higher enzyme expression, resulting in lower response to chemotherapy compared to wild type (87). 

Figure 10-5. Conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase. The importance of folate coenzymes in this reaction is illustrated. NADPH + H provide the necessary reducing equivalents and serine is the source of one-carbon units present on N, N °-methylene tetrahydrofolate (THF).

Site of action 5-FU per se is devoid of antineoplastic activity and must be converted to the corresponding deoxynucleotide (5-FdUMP, Figure 38.9), which competes with deoxyuridine monophosphate (dUMP) for thymidylate synthetase. 5-FdUMP acts as a pseudosubstrate and is entrapped with the enzyme and its N5,N10-methylene tetrahydrofolic acid coenzyme in a ternary complex that cannot proceed to products. DNA synthesis decreases due to lack of thymidine, leading to imbalanced cell growth and cell death. [Note Leucovorin is given with 5-FU because the reduced folate coenzyme is required in the thymidylate synthetase reaction. Lack of sufficient coenzyme reduces the effectiveness of the antipyrimidine.] 5-FU is also incorporated into RNA and low levels have been detected in DNA. 

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. 

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. 

The dUMP Suppression Test Rapidly dividing ceUs can either use preformed TMP or can synthesize it de novo from dUMP. Isolated hone marrow cells or stimulated lymphocytes incubated with pH]TMP wUl 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 pH] TMP as a result of dUution of the pool of labeled material by newly synthesized TMP. 

The cause of megaloblastosis is depressed DNA synthesis, as a result of impaired methylation of dCDP to TDP, catalyzed by thymidylate synthetase, but more or less normal synthesis of RNA. As discussed in Section 10.3.3, thymidylate synthetase uses methylene tetrahydrofolate as the methyl donor it is obvious that folic acid deficiency will result in unpaired thymidylate synthesis. It is less easy to see how vitamin B12 deficiency results in impaired thymidylate synthesis without invoking the methyl folate trap hypothesis (Section 10.3.4.1). The main circulating form of folic acid is methyl-tetrahydrofolate before this can be used for other reactions in tissues, it must be demethylated to yield free folic acid. The only reaction that achieves this is the reaction of methionine synthetase (Section 10.8.1). Thus, vitamin B12 deficiency results in a functional deficiency of folate. 

Scheme III shows the experimental arrangement to study the second one-carbon transfer reaction we investigated, the formation of thymidylic acid from uridylic acid catalyzed by thymidy-late synthetase. In this reaction, the methyl group of thymidy-late is derived from the carbon and the two hydrogens of the methylene bridge plus H-6 of methylene-tetrahydrofolate. To study the stereochemistry of this reaction, we (5) synthesized serine stereospecifically labeled with tritium and deuterium at...

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. 

Several processes described above use one-carbon derivatives of tetrahydrofolic acid (Fig. 14-22). E.g., the synthesis of the purine ring (Eig. 14-18) requires N °-formyl tetrahydrofolate. Thymidylate synthetase, a key enzyme in pyrimidine synthesis, uses FP,N -methylene tetrahydrofolate both as a donor of a methyl group... 

Formation of a covalent adduct with an enzymatic nucleophile increases the reactivity of the substrate, which facilitates a reaction that is distinct from adduct formation. Examples of this include the thymidylate synthetase, in which the sulfhydryl of an active site Cys adds to C6 of the uracil. This breaks the aromaticity and adds electron density to increase the nucleophilicity of C5, thereby facilitating transfer of a methylene equivalent from tetrahydrofolate (Carreras and Santi, 1995). 

Trimethoprim, by blocking the reduction of dihydrofolate, reduces the tetrahydrofolate pool more rapidly. In part, the attrition of the tetrahydrofolate pool is assisted by the activity of thymidylate synthetase, since in this reaction the cofactor not only transfers a one-carbon fragment (methylene) but contributes to the reduction of the methylene group to methyl, and is itself oxidized to dihydrofolate. The effect of trimethoprim is thus to trap folates in the dihydro- state, and the functional tetrahydrofolate pool is depleted. 

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