Dihydrofolic acid reductase reaction

The combination of trimethoprim and sulfamethoxazole (usually five parts sulfamethoxazole to one part trimethoprim) interferes with the synthesis of active folic acid by means of two separate reactions. In the first, sulfonamides compete with PABA and prevent its conversion to dihydro-folic acid. In the second, trimethoprim, by inhibiting the activity of dihydrofolic acid reductase, prevents the conversion of dihydrofolic acid into tetrahydrofolic acid, which is necessary for the synthesis of DNA. These reactions are summarized in Figure 90. 

FIGURE 18.35 Formation of THF from folic acid by the dihydrofolate reductase reaction. The R group on these folate molecules symbolizes the one to seven (or more) glutamate units that folates characteristically contain. All of these glutamates are bound in y-carboxyl amide linkages (as in the folic acid structure shown in the box A Deeper Look Folic Acid, Pterins, and Insect VFingis). The one-carbon units carried by THF are bound at N, or at or as a single carbon attached to both... 

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... 

Aminopterin and amethopterin (methotrexate) are 4-amino analogues of folic acid (Fig. 11.3) and interfere with the production of the active folate coenzyme by blocking the enzyme dihydrofolate reductase (reaction b in Fig. 13.1) (see also Fig. 11.6). 

Fig. 14. Dihydrofolic acid. In the dihydrofolate reductase reaction, the double bond between N-5 and C-6 is reduced by hydride transfer from the 4-pro-R position of NADPH to C-6, and addition of a proton at N-5.

Dihydrofolate reductase (DR) is showm near the top of Figure 9.6, This enzyme catalyzes the reduction of Hjfolate to H folate. DR is part of the cycle of reactions in the synthesis of thymidylic acid. The enzyme also catalyzes the reduction of folic acid to dihydrofolic acid and then to EclrahydrofoJic acid, DR is the target of the anti cancer drug methotrexate (MTX), MTX exerts its toxic effects more nn rapidly... 

In the human, dietary folic acid must be reduced metaboli-cally to THFA to exert its vital biochemical actions. This reaction, which proceeds through the intermediate dihydro-folic acid, is catalyzed by a reductase. This reductase enzyme system has been implicated as the catalyst in both reaction steps—folic acid reduction and dihydrofolic acid reduction. The coenzyme THFA is converted to other cofactors by formulation of the N-IO and/or N-5 nitrogen. 

This enzyme also catalyzes conversion of dihydrofolate (FH2) to tetrahydrofolate (FH4), and folic acid contains a pteridine ring system (see the discussion of one-carbon metabolism in Chapter 27). However, regeneration of tetrahydrobiopterin by the dihydrofolate reductase reaction, however, is too slow to support normal rates of phenylalanine hydroxylation. 

Dihydrofolate reductase (DHFR) (EC 1.5.1.3) catalyzes the NADPH-depen-dent reduction of dihydrofolic acid, thereby restoring THFA as a central cofactor in Cl transfer reactions. Commonly the enzyme has been found to be monomeric, with a molecular mass around 20 kDa. It possesses an a/p fold and does not contain a cofactor. Phylogenetically, the relationship of the enzyme to eukaryotic homologs seems to be closer than to other bacterial DHFRs. Since the expression of the natural enzyme in Tm is extremely low, studies focused on the structure, stability, and folding of the recombinant protein. For its preparation and general characterization, cf. ref. 91a. 

Futterman (47) has partially purified an enzjrme system from chicken liver that reduces folic acid and dihydrofolic acid (FHj) to FH4. TPNH was required for the reduction of FA to FHj, but either TPNH or DPNH were able to bring about the reduction of FHj to FH4. This enzyme dihydrofolic reductase was further purified by Osborn and Huennekens (48) who found that it carried out the rever ble reaction. 

A rare case of enzyme catalyzing imine reduction reaction (see also Section 13.4.4) is the stereoselective reduction of dihydrofolic acid to (6S)-tetrahydrofolic acid by dihydrofolate reductase (DHFR) at the expense of NADPH. This biocatalytic step was employed in the synthesis of (S)-leucovorin [(6S)-5-formyl-5,6,7,8-tetrahydro-folate], a drug used in cancer chemotherapy. DHFR produced by E. coli was combined with a GDH/glucose cofactor recycling system and yielded (6S)-tetrahy-drofolic acid, which upon formylation furnished L-leucovorin with >99.5% de (Figure 13.31) [37-39]. 

The NAD- and NADP-dependent dehydrogenases catalyze at least six different types of reactions simple hydride transfer, deamination of an amino acid to form an a-keto acid, oxidation of /3-hydroxy acids followed by decarboxylation of the /3-keto acid intermediate, oxidation of aldehydes, reduction of isolated double bonds, and the oxidation of carbon-nitrogen bonds (as with dihydrofolate reductase). 

Folates carry one-carbon groups in transfer reactions required for purine and thymidylic acid synthesis. Dihydrofolate reductase is the enzyme responsible for supplying reduced folates intracellularly for thymidylate and purine synthesis. 

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

In the context of preparing potential inhibitors of dihydrofolate reductase (DHFR), the group of Organ has developed a rapid microwave-assisted method for the preparation of biguanide libraries (Scheme 6.174) [330]. Initial optimization work was centered around the acid-catalyzed addition of amines to dicyandiamide. It was discovered that 150 °C was the optimum temperature for reaction rate and product recovery, as heating beyond this point led to decomposition. While the use of hydrochloric acid as catalyst led to varying yields of product, evaluation of trimethylsilyl chloride in acetonitrile as solvent led to improved results. As compared to the protic... 

Pemetrexed is chemically similar to folic acid. It inhibits three enzymes used in purine and pyrimidine synthesis - thymidylate synthetase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase. By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA. In 2004 it was approved for treatment of malignant pleural mesothelioma and as a second-line agent for the treatment of non-small cell lung cancer. Adverse effects include gastrointestinal complaints, bone marrow suppression, alopecia, allergic and neurotoxic reactions. 

The active form of folic acid, tetrahydrofolic acid (THF), is produced from folate by dihydrofolate reductase in a two-step reaction requiring two moles of NADPH. The carbon unit carried by THF is bound to nitrogen N5 or N10, or to both N5 and N10. THF allows one-carbon compounds to be recognized and manipulated by biosynthetic enzymes. Figure 20.11 shows the structures of the various members of the THF family, and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate. 

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... 

The cell-free system prepared from washed embryos has much higher translational activity than the conventional system (compare Fig. 3A and B). When 5 -capped dihydrofolate reductase (DHFR) mRNA containing 549 nt of 3 UTR with a pA tail was incubated with newly prepared as well as conventional extract, there was almost linear kinetics in DHFR synthesis over 4 h, compared with the regular system, which ceased to function after 1.5 h. Further, when washed extract in the reaction volume was increased to 48%, amino acid incorporation occurred initially at a rate twice that of 24% extract, and then stopped after 1 h. However, this pause was caused by a shortage of substrates rather than an irreversible inactivation of ribosomes or factors necessary for translation addition of amino acids, ATP, and GTP after cessation of the reaction (arrow) restarted... 

The active form of folate is the tetrahydro-derivative that is formed through reduction by dihydrofolate reductase. This enzymatic reaction (Figure 29.5) is inhibited by trimethoprim, leading to a decrease in the folate coenzymes for purine, pyrimidine, and amino acid synthesis. Bacterial reductase has a much stronger affinity for trimethoprim than does the mammalian enzyme, which accounts for the drug s selective toxicity. [Note Examples of other folate reductase inhibitors include pyrimethamine, which is used with sulfonamides in parasitic infections (see p. 353), and methotrexate, which is used in cancer chemotherapy (see p. 378).]... 

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. 

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