Oxidation 5-methyl-tetrahydrofolate

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. 

Figure 21-3. The methionine synthase reaction. Methionine synthase catalyzes the remethylation of homocysteine to methionine. In the first half reaction (1), a methyl group is transferred from 5-methyl tetrahydrofolate (5-MTHF) to the reduced form of cobalamin [Cob(I)], generating methyl-cobalamin [Methyl-Cob(III)] and tetrahydrofolate (THF). During the second half reaction (2), the methyl group is transferred from methylcobalamin to homocysteine, generating methionine. During the catalytic reaction, Cob(I) occasionally becomes oxidized, producing an inactive form of cobalamin, cob(II)alamin [Cob(II)]. The enzyme methionine synthase reductase (MTRR) then reactivates Cob(II) through reductive methylation, producing methyl-Cob(III). SAM, 5-adenosylmethionine SAH, 5-adeno-sylhomocysteine.

Methylene-Tetrahydrofolate Reductase The reduction of methylene-tetrahydrofolate to methyl-tetrahydrofolate, shown in Figure 10.7, is catalyzed hy methylene-tetrahydrofolate reductase, a flavin adenine dinucleotide-dependent enzyme during the reaction, the pteridine ring of the substrate is oxidized to dihydrofolate, then reduced to tetrahydrofolate by the flavin, which is reduced by nicotinamide adenine dinucleotide phosphate (NADPH Matthews and Daubner, 1982). The reaction is irreversible under physiological conditions, and methyl-tetrahydrofolate - which is the main form of folate taken up into tissues (Section 10.2.2) - can only be utilized after demethylation catalyzed by methionine synthetase (Section 10.3.4). 

Impairment of methionine synthetase activity, for example, in vitamin B12 deficiency or after prolonged exposure to nitrous oxide (Section 10.9.7), will result in the accumulation of methyl-tetrahydrofolate. This can neither be utilized for any other one-carbon transfer reactions nor demethylated to provide free tetrahydrofolate. 

Experimental animals that have been exposed to ititrous oxide to deplete vitamin B12 show an increase in the proportion of liver folate present as methyl-tetrahydrofolate (85% rather than the normal 45%), largely at the expense of unsubstituted tetrahydrofolate and increased urinary loss of methyl-tetrahydrofolate (Horne et al., 1989). Tissue retention of folate is impaired because methyl-tetrahydrofolate is a poor substrate for polyglutamyl-folate synthetase, compared with unsubstituted tetrahydrofolate (Section 10.2.2.1). As a result of this, vitamin B12 deficiency is frequently accompanied by biochemical evidence of functional folate deficiency, including impaired metabolism of histidine (excretion of formiminoglutamate Section 10.3.1.2) and impaired thymidylate synthetase activity (as shown by abnormally low dUMP suppression Section 10.3.3.3), although plasma concentrations of methyl-tetrahydrofolate are normal or elevated. 

The activity of 10-formyl-tetrahydrofolate dehydrogenase, which catalyzes the oxidation of 10-formyl tetrahydrofolate to CO2 and tetrahydrofolate, is reduced at times of low methionine availability as a means of conserving valuable one-carbon fragments. Therefore, there is no sink for one-carbon substituted tetrahydrofolate, and increasing amounts of folate are trapped as methyl-tetrahydrofolate that cannot be used because of the lack ofvitantin B12 (Krebs etal., 1976). 

Cobalt accepts a methyl group from methyl-tetrahydrofolate, forming methyl Co +-cobalamin. Transfer of the methyl group onto homocysteine results in the formation of Co+-cobalamin, which can accept a methyl group from methyl-tetrahydrofolate to reform methyl Co +-cobalamin. However, except under strictly anaerobic conditions, demethylated Co+-cobalamin is susceptible to oxidation to Co +-cobalamin, which is catalyticaUy inactive. Reactivation of the enzyme requires reductive methylation, with S-adenosyl methionine as the methyl donor, and a flavoprotein linked to NADPH. For this reductive reactivation to occur, the dimethylbenzimidazole group of the coenzyme must be displaced from the cobalt atom by a histidine residue in the enzyme (Ludwig and Matthews, 1997). 

Administration of diphenylhydantoin leads to decreased activity of methylene tetrahydrofolate reductase and an increased rate of oxidation of formyl tetrahydrofolate (increased oxidation of formate and histidine), with a fall in methylene- and methyl-tetrahydrofolate - the reverse of the effect of the methyl folate trap (Billings, 1984a, 1984b). 

In experimental animals and with isolated tissue preparations and organ cultures, the test can be refined by measuring the production of G02 from [ C]histidine in the presence and absence of added methionine. If the impairment of histidine metabolism is the result of primary folate deficiency, the addition of methionine wUl have no effect. By contrast, if the problem is trapping of folate as methyl-tetrahydrofolate, the addition of methionine will restore normal histidine oxidation as a result of restoring the inhibition of methylene-tetrahydrofolate reductase by S-adenosylmethionine and restoring the activity of 10-formyl-tetrahydrofolate dehydrogenase, thus permitting more normal folate metabolism (Section 10.3.4.1). 

Metabolic Role. Riboflavin coenzymes are required for most oxidations of carbon-carbon bonds (Fig. 8.29). Examples include the oxidation of succinyl CoA to fumarate in the Krebs cycle and introduction of a,jS-unsaturation in /3-oxidation of fatty acids. Riboflavin is also required for the metabolism of other vitamins, including the reduction of 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate (Fig. 8.49), and interconversion of pyridoxine-pyridoxal phos-phate-pyridoxamine (Fig. 8.33). Because oxi-dation/reductions that use FAD or FMN as the coenzyme constitute a two-step process, some flavin coenzyme systems contain more than one FAD or FMN. 

On the other hand, dissimilation of acetate may take place by reversal of the pathway used by organisms such as Clostridium thermoace-ticum for the synthesis of acetate from C02. In the degradation of acetate, the pathway involves a dismutation in which the methyl group is successively oxidized via methyl tetrahydrofolate to C02 while the carbonyl group is oxidized via bound carbon monoxide. Such THF-mediated reactions are of great importance in the anaerobic degradation of purines that will be discussed in Section 6.7.4.I. 

Reaction 12 of Figure 34—7 is the only reaction of pyrimidine nucleotide biosynthesis that requires a tetrahydrofo-late derivative. The methylene group of A A Tmethyl-ene-tetrahydrofolate is reduced to the methyl group that is transferred, and tetrahydrofolate is oxidized to dihydro-... 

The best characterized B 12-dependent methyltransferases is methionine synthase (Figure 15.11) from E. coli, which catalyses the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form methionine and tetrahydrofolate. During the catalytic cycle, B12 cycles between CH3-Co(in) and Co(I). However, from time to time, Co(I) undergoes oxidative inactivation to Co(II), which requires reductive activation. During this process, the methyl donor is S-adenosylmethionine (AdoMet) and the electron donor is flavodoxin (Fid) in E. coli, or methionine synthase reductase (MSR) in humans. Methionine synthase... 

One-carbon units in different oxidation states are required in the pathways producing purines, thymidine, and many other compounds. When a biochemical reaction requires a methyl group (methylation), S-adenos dmethionme (SAM) is generally the methyl donor. If a one-carbon unit in another oxidation state is required (methylene, methenyl, formyl), tetrahydrofolate (THF) typically serves as its donor. 

The coenzyme tetrahydrofolate (THF) is the main agent by which Ci fragments are transferred in the metabolism. THF can bind this type of group in various oxidation states and pass it on (see p. 108). In addition, there is activated methyl, in the form of S-adenosyl methionine (SAM). SAM is involved in many methylation reactions—e. g., in creatine synthesis (see p. 336), the conversion of norepinephrine into epinephrine (see p. 352), the inactivation of norepinephrine by methylation of a phenolic OH group (see p. 316), and in the formation of the active form of the cytostatic drug 6-mercaptopurine (see p. 402). 

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. 

Folate is a relatively unstable nutrient processing and storage conditions that promote oxidation are of particular concern since some of the forms of folate found in foods are easily oxidized. The reduced forms of folate (dihydro- and tetrahydrofolate) are oxidized to p-aminobenzoylglutamic acid and pterin-6-carboxylic acid, with a concomitant loss in vitamin activity. 5-Methyl-H4 folate can also be oxidized. Antioxidants (particularly ascorbic acid in the context of milk) can protect folate against destruction. The rate of the oxidative degradation of folate in foods depends on the derivative present and the food itself, particularly its pH, buffering capacity and concentration of catalytic trace elements and antioxidants. 

Figure 2. Aerobic catabolism of methylated sulfides (adapted from Kelly, 1988). 1) DMSO reductase (Hyphomicrobium sp.) 2) DMDS reductase (Thiobacillus sp. 3) trimethylsulfonium-tetrahydrofolate methyltransferase (Pseudomonas sp.) 4) DMS monooxygenase 5) methanethiol oxidase 6) sulfide oxidizing enzymes 7) catalase 8) formaldehyde dehydrogenase 9) formate dehydrogenase 10) Calvin cycle for CO2 assimilation (Thiobacillus sp.) 11) serine pathway for carbon assimilation (Hyphomicrobium sp.).

The donation of the methyl group from N5,N10-methylene tetrahydrofolate leads to the oxidation of the cofactor to dihydrofolate. This points to the importance of dihydrofolate reductase (DHFR) in the functioning of thymidylate synthase. Thus, synthesis of TMP requires a supply of both methyl groups—for example, from serine— and reducing equivalents. 

Tetrahydrofolate functions as a carrier of one-carbon units. There are numerous metabolic reactions that require either the addition or removal of a one-carbon unit of some specific oxidation state. THF binds one-carbon units of three oxidation levels the methanol, formaldehyde, and formate states. These are shown in Table 6.4 along with their origins and uses. The various one-carbon units are interconvertible, as shown in Figure 6.5. Nicotinamide coenzymes are involved. In addition, the one-carbon unit may be released as C02. The methanol-level THF-bound one-carbon unit 5-methyl-THF is the storage and transport form. Once formed, its main pathway of metabolism is to form methionine from homocysteine, a reaction that requires vitamin B12 in the form of methylcobalamin (see Figure 6.2 and Chapter 20) ... 

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. 

Cells making DNA must also be able to make deoxythymidine triphosphate (dTTP). The key step in the synthesis of dTTP is the conversion of dUMP to dTMP via thymidylate synthase. The reaction requires a source of N5,Nw-methylene tetrahydrofolate (see Sec. 15.7, Fig. 15-19) to provide the methyl group. In this reaction, the tetrahydrofolate is oxidized to dihydrofolate. Dihydrofolate must be reduced to tetrahydrofolate via the enzyme dihydrofolate reductase so that more Af5,A,l0-methylene tetrahydrofolate can be made from serine in a reaction catalyzed by serine hydroxymethyltransferase. These three reactions, which are essential for the formation of dTMP, are shown below. 

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