Tetrahydrofolate, dihydrofolate reduction

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). 

Catalytic reduction of folic acid to 5,6,7,8-tetrahydrofolic acid (225) proceeds fast in trifluoroacetic acid (66HCA875), but a modified method using chemical reductants leads with sodium dithionite to 7,8-dihydrofolic acid (224). Further treatment with sodium borohydride gives (225) which has been converted into 5-formyl-(6i ,S)-5,6,7,8-tetrahydro-L-folic acid (leucovorin) (226) by reaction with methyl formate (equation 70) (80HCA2554). 

Folic acid derivatives (folates) are acceptors and donors of one-carbon units for all oxidation levels of carbon except that of CO2 (where biotin is the relevant carrier). The active coenzyme form of folic acid is tetrahydrofolate (THF). THF is formed via two successive reductions of folate by dihydrofolate reductase (Figure 18.35). One-carbon units in three different oxidation states may be bound to tetrahydrofolate at the and/or nitrogens (Table 18.6). These one-carbon units... 

Folic acid antagonist inhibits dihydrofolate reductase (DHFR) blocks reduction of folate to tetrahydrofolate inhibits de novo purine synthesis results in arrest of DNA, RNA, and protein synthesis... 

The dihydrofolate reductase enzyme (DHFR) is involved in one-carbon metabolism and is required for the survival of prokaryotic and eukaryotic cells. The enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is required for the biosynthesis of serine, methionine, purines, and thymidylate. The mouse dihydrofolate reductase (mDHFR) is a small (21 kD), monomeric enzyme that is highly homologous to the E. coli enzyme (29% identify) (Pelletier et al., 1998). The three-dimensional structure of DHFR indicates that it is comprised of three structural fragments F[l], F[2] andF[3] (Gegg etal., 1997). 

DHFR has been the object of intense research for the last few decades. The enzyme catalyses the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8 tetrahydrofolate, a chemical which participates in the thymidilate synthesis cycle. Thus, the enzyme is crucial in the synthesis of thymidine monophosphate as well as in various one-carbon unit transfer reactions. 

The reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) has been analyzed extensively14 26-30 and a kinetic scheme for E. Coli DHFR was proposed in which the steady-state kinetic parameters as well as the full time course kinetics under a variety of substrate concentrations and pHs were determined. From these studies, the pKa of Asp27 is 6.5 in the ternary complex between the enzyme, the cofactor NADPH and the substrate dihydrofolate. The second observation is that, contrary to earlier results,27 the rate determining step involves dissociation of the product from the enzyme, rather than hydride ion transfer from the cofactor to the substrate. 

In this study, identification of the critical atomic and molecular determinants pertaining to the mechanism of dihydrofolate to tetrahydrofolate reduction was achieved by (i) ab initio quantum mechanics, (ii) molecular mechanics, and (iii) free energy perturbation techniques. For the first time, the complete free energy profile was calculated for the proton transfer from Asp27 of the enzyme E. Coli DHFR to the N5 position of the dihydropterin moiety of the substrate dihydrofolate. In addition, the free... 

In a collaboration between the Abelson and Hecht labs [56b], a series of noncoded amino acids were introduced into dihydrofolate reductase (DHFR) to probe substrate binding and the requirement of an aspartic acid residue for catalytic competence. When aspartic acid analogs mono- or disubstituted at the )0-carbon were substituted for the active site aspartic acid residue, the mutant DHFRs were still able to catalyze the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate at 74 - 86 % of the wild-type rate. While hydride transfer from NADPH is not the rate-limiting step for the wild-type enzyme at physiological pH, a kinetic isotope experiment with NADPD indicated that hydride transfer had likely become the rate-limiting step for the mutant containing the )0,)0-dimethylaspartic acid. 

Folic acid becomes sequentially reduced in the body by the enzyme dihydrofolate reductase to give dihydrofolic acid (FH2) and then tetrahydrofolic acid (FFi4). Reduction occurs in the pyrazine ring portion. 

Thymidylate synthase [EC 2.1.1.45] reductively methylates 2 -deoxyuridine-5 -monophosphate to form 2 -deoxythymidine-5 -monophosphate in the following folate-dependent reaction dUMP + A, A -methylene-tetrahydrofolate dTMP + dihydrofolate. 

The donor coenzyme for the one-carbon transfer is AT, Ar °-methylene tetrahydrofolate (A A °-methylene THF) simultaneous reduction to a methyl group leaves dihydrofolate (DHF) as byproduct. 

I I 3. The answer is c. (Hardman, pp 1243-1247.) Antimetabolites of folic acid such as methotrexate, which is an important cancer chemotherapeutic agent, exert their effect by inhibiting the catalytic activity of the enzyme dihydrofolate reductase. The enzyme functions to keep folic acid in a reduced state. The first step in the reaction is the reduction of folic acid to 7,8-dihydrofolic acid (FH2), which requires the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). The second step is the conversion of FH2 to 5,6,7,8-tetrahydrofolic acid (FH ). This part of the reduction reaction requires nicotinamide adenine dinucleotide (NADH) or NADPH. The reduced forms of folic acid are involved in one-carbon transfer reactions that are required during the synthesis of purines and pyrimidine thymidylate. The affinity of methotrexate for dihydrofolate reductase is much greater than for the substrates of folic acid and FH2. The action of... 

Conversion of dUMP to dTMP is catalyzed by thy-midylate synthase. A one-carbon unit at the hydroxymethyl (—CH2OH) oxidation level (see Fig. 18-17) is transferred from Af5,Af10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group (Fig. 22-44). The reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate, which is unusual in tetrahydrofolate-requiring reactions. (The mechanism of this reaction is shown in Fig. 22-50.) The dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase—a regeneration that is essential for the many processes that require tetrahydrofolate. In plants and at least one protist, thymidylate synthase and dihy-drofolate reductase reside on a single bifunctional protein. 

Methotrexate and related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, catalyzed by dihydrofolate reductase (see p. 371). 

Another group of inhibitors prevents nucleotide biosynthesis indirectly by depleting the level of intracellular tetrahydrofolate derivatives. Sulfonamides are structural analogs of p-aminobenzoic acid (fig. 23.19), and they competitively inhibit the bacterial biosynthesis of folic acid at a step in which p-aminobenzoic acid is incorporated into folic acid. Sulfonamides are widely used in medicine because they inhibit growth of many bacteria. When cultures of susceptible bacteria are treated with sulfonamides, they accumulate 4-carboxamide-5-aminoimidazole in the medium, because of a lack of 10-formyltetrahydrofolate for the penultimate step in the pathway to IMP (see fig. 23.10). Methotrexate, and a number of related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. These inhibitors are structural analogs of folic acid (see fig. 23.19) and bind at the catalytic site of dihydrofolate reductase, an enzyme catalyzing one of the steps in the cycle of reactions involved in thymidylate synthesis (see fig. 23.16). These inhibitors therefore prevent synthesis of thymidylate in replicating... 

Methotrexate acts by inhibition of dihydrofolate reductase, the enzyme requisite for the reduction of dihydrofolic acid (3) to 5,6,7,8-tetrahydrofolic acid (4). In turn, (4) is a precursor to a series of enzyme cofactors (5-7) essential for the transfer of one carbon unit necessary for the biosynthesis of purines and pyrimidines and hence, ultimately, DNA. As an inhibitor of dihydrofolate reductase, methotrexate kills cells during the S phase of the cell cycle, when the cells are in the log phase of growth. Unfortunately, this cytotoxicity is non-selective, and rapidly proliferating normal cells, e.g., gastrointestinal epithelium cells and bone marrow, are dramatically affected as well. In addition, recent use of high dose methotrexate therapy with leucovorin rescue has led to additional clinical problems arising from a dose-related nephrotoxic metabolite, 7-hydroxy methotrexate (8). Finally, the very polar nature of methotrexate renders it virtually impenetrable to the blood-brain barrier, which can necessitate direct intrathecal injection in order to achieve therapeutic doses for the treatment of CNS tumours. 

Methotrexate and aminopterin are believed to inhibit dihydrofolate reductase by blocking the reduction of dihydrofolate to tetrahydrofolate, which is the cofactor in the synthesis of thymine and purines used in DNA synthesis. 

Aminopterin and amethopterin are 4-amino analogues of folic acid (Fig. 11.5) and as such are potent inhibitors of the enzyme dihydrofolate reductase (EC 1.5.1.3) (Blakley, 1969). This enzyme catalyses the reduction of folic acid and dihydrofolic acid to tetrahy-drofolic acid which is the level of reduction of the active coenzyme involved in many different aspects of single carbon transfer. As is clear from Fig. 11.6, tetrahydrofolate is involved in the metabolism of (a) the amino acids glycine and methionine (b) the carbon atoms at positions 2 and 8 of the purine ring (c) the methyl group of thymidine and (d) indirectly in the synthesis of choline and histidine. 

Folate in the human organism is converted to tetrahydrofolate (THF or FH4) by a reductive process. The reduction reaction is a stepwise one folate to dihydrofolate, then to THF. A single enzyme, dihydrofolate reductase, catalyzes both steps. The reaction is inhibited by folate analogues and the antitumor agents methotrexate and aminopterin (Figure 6.3). Because THF is required for DNA biosynthesis (Chapter 10) and tumors have a very high level of DNA biosynthetic activity, even modest decreases in THF availability will inhibit tumor growth. 

The synergistic antimicrobial activity of co-trimoxazole results from its inhibition of two sequential steps in the synthesis of tetrahydro-folic acid sulfamethoxazole inhibits the incorporation of PABA into folic acid, and trimethoprim prevents reduction of dihydrofolate to tetrahydrofolate (see Figure 29.5). Co-trimoxazole exhibits more potent antimicrobial activity than sulfamethoxazole or trimethoprim alone (seed Figure 29.6). 

The two steps in the reduction of folic acid to tetrahydrofolate are catalyzed by dihydrofolate reductase. Both of these reactions require NADPH as a source of electrons. 

The next step in folic acid synthesis is the reduction of dihydrofolate to tetrahydrofolate. This can be done by both humans and bacteria and, although it looks like a rather trivial reaction (see black portion of molecules), it can only be done by the very important enzyme dihydrofolate reductase. 

This is a small monomeric enzyme (molecular weight around 20000) that catalyses reduction of a double bond between carbon and nitrogen, converting 7,8-dihydrofolate into 5,6,7,8-tetrahydrofolate (Fig. 14). Hydride is transferred from the 4-pro-R position of NADPH to C-6, N-5 acquiring a proton [64-66]. 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

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