Interconversion of Substituted Folates

Methylene-, methenyl-, and 10-formyl-tetrahydrofolates are freely interconvertible. The two activities involved - methylene-tetrahydrofolate dehydrogenase and methenyl-tetrahydrofolate cyclohydrolase - form a trifunctional enzyme with 10-formyl-tetrahydrofolate synthetase (Paukert et al., 1976). This means that single-carbon fragments entering the folate pool in any form other than as methyl-tetrahydrofolate can be readily available for any of the biosyn-thetic reactions shown in Figure 10.4.

The conversion of 5-formyl-tetrahydrofolate to methenyl-tetrahydrofolate, catalyzed by 5-formyl-tetrahydrofolate cyclohydrolase, is important. Although 5-formyl-tetrahydrofolate is the most commonly used pharmaceutical preparation of the vitamin, a relatively large proportion of orally administered

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

5-formyl-tetrahydrofolate undergoes nonenzymic cyclization to methenyl-tetrahydrofolate in the acid conditions of the stomach. Methylene-Tetrahydrofolate Reductase The reduction of methylene-tetrahydrofolate to methyl-tetrahydrofolate, shown in Figure 10.7, is catalyzed by methylene-tetrahydrofolate reductase, a flavin adenine di-nucleotide-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).

Methylene-tetrahydrofolate reductase is inhibited by S-adenosylmethi-onine, which inhibits reduction of the flavin prosthetic group by NADPH. S-Adenosylhomocysteine overcomes this inhibition to some extent, as might be expected for an enzyme that is indirectly involved in the regulation of methionine and S-adenosylmethionine concentrations in the cell.

Kang and coworkers (1991) reported a variant of methylene-tetrahydrofo-late reductase, in which cytosine677 is replaced by thymidine, resulting in a change of alanine226 to valine, in people who were hyperhomocysteinemic (Section The variant enzyme is thermolabile (i.e., it is unstable to heating to about 40° to 45°C), and subjects who are homozygous for the thermolabile enzyme have about 50% of normal enzyme activity in tissues. Not only is the enzyme labile on moderate heating in vitro, but it is also unstable in vivo. There are considerable differences in the frequency of the variant gene in differentpopulation groups, ranging from 1.2% ofBrazilian Amerindians, 3.1% of British South Asians, and 10% of British and Australian white people, with upto 30% of Italians and 35% of Japanese being homozygous for the thermola-bile variant. Some studies have shown that the thermolabile variant was two-to three-fold more common among people with atherosclerosis and coronary heart disease than among disease-free people of the same ethnic origin.

Being homozygous for the thermolabile variant of methylene-tetrahydro-folate reductase is a necessary, but not sufficient, condition for the development of hyperhomocysteinemia. Homozygotes with a high folate intake have plasma concentrations of homocysteine as low as heterozygotes or people who are homozygous for the normal (stable) form of the enzyme (Jacques et al., 1996). Two possible mechanisms have been proposed to explain how a relatively high intake of folate can mask the effect of being homozygous for the thermolabile variant of methylene-tetrahydrofolate reductase:

1. Most dietary folate is reduced and methylated to methyl-tetrahydro-folate in the intestinal mucosa (Section 10.2.1). Intestinal mucosal cells have a rapid turnover, typically 48 hours from proliferation in the crypt to shedding at the tip of the villus. This means that an unstable variant of the enzyme, which loses activity over a shorter time than the normal enzyme, is probably irrelevant in cells that have such a rapid turnover. A high intake of folate would therefore result in a relatively high rate of supply of methyl-tetrahydrofolate to cells, arising from newly absorbed folate, so that impaired turnover of folate within cells would be less important.

2. In common with a number of enzymes, methylene-tetrahydrofolate reductase may be more resistant to thermal denaturation (and hence possibly more stable) in the presence of its substrate. Hence it is possible that high tissue levels of methylene-tetrahydrofolate (resulting from a high folate status) may protect the enzyme and enhance its stability (Guenther et al., 1999; Yamada et al., 2001a). However, it is unlikely that a high intake of folate would lead to a sufficient accumulation of methylene-tetrahydrofolate to stabilize the enzyme in this way because, as discussed in Section, there is rapid interconversion between the various one-carbon substituted folates. Any excess is converted to formyl-tetrahydrofolate, then the formyl group is oxidized to carbon dioxide to maintain a pool of free folate for the collection of one-carbon fragments in catabolic reactions.

Methylene-tetrahydrofolate reductase is a flavoprotein. There is some evidence that riboflavin also stabilizes the thermolabile variant and that riboflavin supplements may lower plasma homocysteine in people who are homozygous for the variant enzyme (McNulty et al., 2002).

There is a second polymorphism of methylene-tetrahydrofolate reductase in about 10% of the population, in which adenosine 1298 is replaced by cytosine. Like the thermolabile variant, this results in about 50% of normal activity of the enzyme in lymphocytes from homozygotes and the development of hy-perhomocysteinemia which, in this case, does not seem to be responsive to high intakes of folate (Chango et al., 2000a, 2000b). Disposal of Surplus One-Carbon Fragments With the exception of serine hydroxymethyltransferase (Section, all of the reactions shown in Figure 10.4 as sources of one-carbon substituted folates are essentially catabolic reactions. When there is a greater entry of single carbon units into the folate pool than is required for biosynthetic reactions, the surplus can be oxidized to carbon dioxide byway of 10-formyl-tetrahydrofolate, thus ensuring the availability of tetrahydrofolate for catabolic reactions.

10-Formyl-tetrahydrofolate dehydrogenase has a high Km relative to the normal intracellular concentration of its substrate and only has significant activity when there is a relative excess of one-carbon substituted tetrahydrofolate. The product, free tetrahydrofolate, is a poor leaving group, and much remains bound to the enzyme, resulting in significant inhibition. The activity of the dehydrogenase is thus strictly regulated by the ratio of formyl-tetrahydrofolate:free tetrahydrofolate in the tissue (Min et al., 1988).

There is an alternative pathway disposal of surplus one-carbon fragments, in which glycine N-methyltransferase catalyzes the methylation of glycine to sarcosine, using S-adenosylmethionine (Section 10.3.4) as the methyl donor. The resultant S-adenosylhomocysteine is then remethylated at the expense of methyl-tetrahydrofolate, thus regenerating free tetrahydrofolate. Factors such as vitamin A-which induce the synthesis of glycine N-methyltransferase-may act to reduce the tissue pool of one-carbon substituted folates for methylation reactions, leading to undermethylation of DNA and possible cancer development (Rowling et al., 2002).

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