Methotrexate, enzymic oxidation

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described above in the section on vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N 5,N 10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33-2, reaction 2). Unlike all of the other enzymatic reactions that utilize folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, one mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate can be consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N 5,N 10-methylenetetrahydrofolate by the action of serine transhydroxy- methylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are often referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 55 Cancer Chemotherapy). 

Dihydrofolate reductase activates folate to tetrahydrofolate with dihydrofolate as an intermediate. Methotrexate, an antitumor agent, inhibits this enzyme. The 5-methyl group is first oxidized to the formaldehyde level, then to the formate level, then to C02. Three steps require three molecules of NAD or an equivalent, for a total of 3 x 3 = 9 ATP molecules. 

Neither methotrexate nor its microbial breakdown product, APA, is a substrate for xanthine oxidase [6, 215], although this may be more a function of the 2,4-diaminopteridine moiety, which itself is refractory to xanthine oxidase [204], rather than the glutamic acid residue. In fact, methotrexate is a potent competitive inhibitor of this enzyme, with a K value of around 25 fiM [218,219]. There is considerable controversy as to whether folic acid, a substituted 2-aminopteridin-4-one, is also an inhibitor of xanthine oxidase. It is not oxidized at carbon 7, unlike the parent compound, which is a poor substrate [204]. However, some workers have shown that folic acid is an extremely potent competitive inhibitor of xanthine oxidase, some 10-times more effective in vitro than allopurinol, whereas other reports claim that the inhibition is due to the contaminant 2-amino-4-oxopteridine-6-aldehyde (27), which is a photolytic breakdown product of folic acid [4, 171, 172, 218-220]. 

Methotrexate inhibits dihydrofolate reductase, an enzyme which reduces dihydrofolate (FH ) to tetrahydrofolate (FH ). Normally, the coenzyme FH transfers a methyl group to dUMP, forming thymidylate, and is oxidized to FH in the process. Methotrexate prevents recycling of this cofactor. Because thymidylate is necessary for DNA synthesis, methotrexate is toxic in the S-phase (synthesis). 

Folic Acid Antagonists - Interest in antimetabolites that interfere with the synthesis of nucleic acids continues. Studies on the transport and uptake of methotrgxate ° and 2,4-diamino-5-(3,4-dichlorophenyl)-6-methylpyrimidine indicate that clinical response is related to cellular uptake of drugl and the resistance of certain cells to methotrexate appears to be due to lack of transport into these cells. Work on other resistant cell lines indicates that resistance can also be due to an increase in cellular content of folate reductase, but no correlation was observed between resistance and the level of other enzymes involved in folate metabolism. Despite these results there is evidence that the ability of methotrexate to kill cells cannot be entirely explained by its inhibition of folate reductase. Leucovorin at appropriately timed intervals improved the therapeutic index of methotrexate in the treatment of head and neck cancer, and lymphosarcoma and reticulum cell sarcoma. The use of methotrexate in the treatment of hormone-refractory metastatic breast carcinoma " and the use of intrathecal methotrexate also appear promising. Oxidation of methotrexate to 7-hydroxymethotrexate by liver aldehyde oxidase is probably a detoxification mechanism. Material previously reported to be tetrahydromethotrexate has now been found to be a mixture of di- and tetrahydromethotrexate, both of which are less effective than methotrexate in the inhibition of folate reductase, but more effective in the inhibition of thymidylate synthetase. ... 

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