Methylene THF reductase

Mammalian methylene-THF reductase is a FAD-containing flavoprotein that utilizes NADPH for the reduction to 5-methyl-THF.427,428 Matthews429 suggested that the mechanism of this reaction involves an internal oxidation-reduction reaction that generates a 5-methyl-quinonoid dihydro-THF (Eq. 15-45). Methylene-THF reductase of acetogenic bacteria is also a flavoprotein but it contains Fe-S centers as well. The 237-kDa a4P4 oligomer contains two molecules of FAD and four to six of both Fe and S2 ions.430 431... 

These Cl groups may be attached to N atoms in positions 5 or 10 (Fig. 14-23) or may form a bridge between the two. A -Methyl THF is formed in mammals by a virtually irreversible reaction that is catalyzed by the enzyme methylene THF reductase, the other THF derivatives are interconverted through a series of oxidation-reduction and hydration-dehydration reactions. 

Rundles and Brewer (1958) have shown that methionine aggrevates bone marrow megaloblastosis and its addition to bone marrow cultures from vitamin deficient patients does not alleviate defective DNA synthesis (Waxman, 1969 Metz, 1968). This antifolate" effect of methionine in bone marrow cultures is in contrast to the "pro-folate effect described in rat liver. A species difference s not involved because similar results were found in rat bone marrow cultures (Cheng et al., 1975) leading to the hypothesis that methionine exerts its primary effect as an end-product inhibitor of the homocysteinetransmethylase reaction rather than regulation (via SAM) of 5 10 methylene THF reductase. It is also possible that the tissue culture system used to study bone marrow metabolism does not reflect the normal cellular environment. As Krebs et al. (1976) have pointed out, isolated normal hepatocytes have lost low molecular weight constituents including methionine and are incapable of some reactions known to occur in vivo. 

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.)...

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

Figure 10.7. Reaction of methylene-tetrahydrofolate reductase (EC 1.7.99.5). THF, tetrahydrofolate.

The deoxyuridylate (dUMP) produced by dephosphorylation of the dUDP product of ribonucleotide reductase is not a component of DNA, but its methylated derivative deoxythymidylate (dTMP) is. The methylation of dUMP is catalyzed by thymidylate synthase, which utilizes N5,N10-methylene THF. As the methylene group is transferred, it is reduced to a methyl group, while the folate coenzyme is oxidized to form dihydrofolate. THF is regenerated from dihydrofolate by dihydrofolate reductase and NADPH. (This reaction is the site of action of some anticancer drugs, such as methotrexate.) Deoxyuridylate can also be synthesized from dCMP by deoxycytidylate deaminase. 

Methotrexate is a close strnctnral analogne of folate and inhibits dihydrofolate reductase. This prevents the rednction of folate and dihydrofolate (DHF) to tetrahydrofolate (THF), which is the precursor of A ,lV -methylene THF. This is essential for dTTP and DNA synthesis. Unfortnnately, normal cells are also attacked by methotrexate. Folinic acid (A -formyl tetrahydrofolate) is an active form of folate that can be given after the start of methotrexate treatment to rescue normal cells from this drug toxicity. 

Notice that the coenzyme that transfers the methylene group to the substrate is also the reagent that subsequently reduces the methylene group to a methyl group. Because the coenzyme is the reducing agent, it is simultaneously oxidized to dihydrofolate. (Recall that oxidation decreases the number of C—H bonds.) Dihydrofolate must then be reduced back to methylene-THF by the enzyme dihydrofolate reductase so that tetrahydrofolate continues to be available as a coenzyme. 

The other major class of antimalarials are the folate synthesis antagonists. There is a considerable difference in the drug sensitivity and affinity of dihydrofolate reductase enzyme (DHFR) between humans and the Plasmodium parasite. The parasite can therefore be eliminated successfully without excessive toxic effects to the human host. DHFR inhibitors block the reaction that transforms deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) at the end of the pyrimidine-synthetic pathway. This reaction, a methylation, requires N °-methylene-tetrahydrofolate as a carbon carrier, which is oxidized to dihydrofolate. If the dihydrofolate cannot then be reduced back to tetrahydrofolate (THF), this essential step in DNA synthesis will come to a standstill. 

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