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Letter to the Editor |
Department of Nutrition Sciences University of Alabama at Birmingham Birmingham, AL 35294 E-mail: matlocks{at}uab.edu
Dear Sir:
The conclusion by Gregory et al (1) in a recent issue of the Journal that "the appearance of both [2H1]- and [2H2]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation" and the statement in an accompanying editorial by Cook (2) that "these results are the first to provide in vivo proof that hepatic one-carbon units derived from serine by mSHMT [mitochondrial serine hydroxymethyltransferase] can be assimilated in the cytosol and used for homocysteine remethylation" deserve further comments. Gregory et al assert that the biosynthesis of [2H1]methionine indicates that folate-dependent one-carbon metabolism of [2,3,3-2H3]serine occurs in the mitochondria. However, [2,3,3-2H3]serine is a poor choice of substrate because enzyme-catalyzed reactions produce isotope exchange that may give similar results without involving folate-dependent one-carbon metabolism in the mitochondria.
For example, reactions catalyzed by serine dehydratase or enzymes in the transamination pathway of serine metabolism result in the exchange of the 2H atom on the 2-position of serine and the formation of deuterated pyruvate. Pyruvate is converted to oxaloacetate by pyruvate carboxylase and is subsequently metabolized to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (pyrophosphate) (3). In the pyruvate carboxylase reaction, there is a two-thirds chance that one of the original 2H atoms on the 3-position of serine is exchanged. Phosphoenolpyruvate is in equilibrium with 2-phosphoglycerate, which is in equilibrium with 3-phosphoglycerate (3). 3-Phosphoglycerate can readily be metabolized back to serine, which is likely to be depleted in 2H at the 3-position. The conversion of pyruvate to oxaloacetate to phosphoenolpyruvate and back to pyruvate is a well known substrate cycle in glycolysis and gluconeogenesis that, because of its quintessential importance, is active in the liver, as are the other steps in carbohydrate metabolism (3). Thus, the above enzymatic reactions could lead to the production of both [3,3-2H2]- and [3-2H1]serine. These isotopes of serine could therefore supply the one-carbon units for folate-dependent [2H2]- and [2H1]methionine biosynthesis. No folate-dependent one-carbon metabolism in the mitochondria is necessary to explain the results of Gregory et al.
In addition, cytosolic 5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase (NADP+) catalyzes the reversible oxidation of 5,10-CH2-THF to 5,10-methenyltetrahydrofolate (5,10-CH=THF) in concert with the reduction of NADP to NADPH (4). Thus, 5,10-C2H2-THF exchanges one 2H, forming NADP2H and 5,10-C2H=THF. On the reverse reaction, NADPH could be used to form 5,10-CH2H-THF. Alternatively, NADP2H could be used to form 5,10-CH2H-THF from 5,10-CH=THF. Both 5,10-CH2H-THF and 5,10-C2H2-THF could then furnish a one-carbon unit for the synthesis of [2H1]- and [2H2]methionine, respectively. Again, no folate-dependent one-carbon metabolism in the mitochondria is required to explain the results of Gregory et al.
Furthermore, NADP2H generated by the above isotope exchange may be used by cytosolic 5,10-CH2-THF reductase to form 5-methyltetrahydrofolate (5-CH22H-THF) from 5,10-CH2-THF (4). 5-CH22H-THF could be used for the biosynthesis of [2H1]methionine.
The reported absence of [2H1]glycine suggests that metabolism of serine occurred by the dehydratase, via the transaminase pathway, or through cysteine (via cystathionine). The rapid appearance of [2H2]- and [2H1]serine also suggests isotope exchange through the above pathways or through 5,10-CH2-THF dehydrogenase and the synthesis of serine from glycine.
Layered on top of the already complex problem of interpreting the data is the possibility of kinetic deuterium isotope effects when any C–2H bond is broken. For example, the kinetic deuterium isotope effect reported for human 5,10-CH2-THF dehydrogenase is 2.9 (5). Deriving correction factors for these isotope effects in vivo is a formidable, if not impossible, task.
Finally, the importance of the mitochondrial folate metabolic pathway shown in Figures 1 and 5 of the article by Gregory et al has been questioned. In mammals, the bifunctional (not trifunctional) enzyme (5,10-CH2-THF dehydrogenase/5,10-CH=THF cyclohydrolase) is found in mitochondria, but at low concentrations. Thus, the formation of formate by the formate–tetrahydrofolate ligase (10-formyltetrahydrofolate synthetase) in mammalian mitochondria has not been established (reference 6 and references therein).
In my opinion, interpreting the data from the experiments reported by Gregory et al is complex and labyrinthine. Until all of the above concerns can be addressed, the data neither support nor refute mitochondrial folate-dependent metabolism as a major source of one-carbon units in human methionine biosynthesis.
REFERENCES
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