Substrate flux through methylenetetrahydrofolate dehydrogenase: predicted effects of the concentration of methylenetetrahydrofolate on its partitioning into pathways leading to nucleotide biosynthesis or methionine regeneration

Abstract

Folic acid exists in mammalian cells with a poly-gamma-glutamate tail that may regulate the flux of folates through the various cellular pathways. The substrate polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver has been examined by using a competitive method and measuring apparent tritium kinetic isotope effects on Vmax/Km for methylenetetrahydrofolate. This competitive method yields very accurate ratios of Km values for alternate substrates of an enzyme and may also be applied to reactions with no isotope effect. In combination with published data from our own and other laboratories, the kinetic parameters of methylenetetrahydrofolate dehydrogenase were used to calculate the initial velocities of pig liver methylenetetrahydrofolate dehydrogenase, thymidylate synthase, and methylenetetrahydrofolate reductase, at physiological concentrations of substrates and enzymes. These calculations suggest that the cellular concentration of methylenetetrahydrofolate may regulate the flux of this metabolite into the pathways leading to nucleotide biosynthesis and methionine regeneration. An increase in the cellular level of methylenetetrahydrofolate would permit more one-carbon units to be directed toward nucleotide biosynthesis.

The Many Flavors of Hyperhomocyst(e)inemia: Insights from Transgenic and Inhibitor-Based Mouse Models of Disrupted One-Carbon Metabolism

“…While it might be thought that folate deficiency would affect all components of the folate pool equally, studies on the in vitro catalytic properties of the enzymes that compete for methylenetetrahydrofolate have suggested that methylenetetrahydrofolate reductase has a much lower Michaelis constant (K m ) for this substrate than thymidylate synthase or methylenetetrahydrofolate dehydrogenase (21), which would be expected to result in methyl trapping. Thus embryos with folate deficiency and those with reduced methionine synthase activity may share a propensity for apoptosis due to insufficient supplies of 10-formyltetrahydrofolate and methylenetetrahydrofolate.…”

The crystal structure of a bacterial, bifunctional 5, 10 methylene‐tetrahydrofolate dehydrogenase/cyclohydrolase

“…Kinetic studies suggest that interaction of the poly-g-glutamyl tail of the methylene-tetrahydrofolate molecule with the enzyme provides binding energy and affinity for the substrate, while allowing dynamic motion and kinetic channeling of the reactive methenylpterin ring system between dehydrogenase and cyclohydrolase active sites~Cohen & MacKenzie, 1978;Wasserman et al, 1983;Green et al, 1988;Hum & MacKenzie, 1991;Pelletier & MacKenzie, 1995!. Inspection of the surface of the E. coli dph0cyc revealed that there are two significant regions of positive-charged residues, one at each end of the cleft between the two a0b domains of deh0cyc.…”

Evidence for Small Ubiquitin-like Modifier-dependent Nuclear Import of the Thymidylate Biosynthesis Pathway

“…The MTHFR-catalyzed reaction commits one-carbon units to the methionine cycle ( Fig. 1) (13). Both reactions are essentially irreversible in vivo (10).…”

“…Metabolic labeling studies, in vitro binding experiments, and mathematical modeling have indicated that methylene-THF is preferentially directed toward methionine synthesis relative to thymi-* This work was supported by Public Health Service Grant DK58144. The costs dylate biosynthesis (13,14). There is increasing evidence that the partitioning of methylene-THF between methionine and thymidylate synthesis is central to the origin of folate-related pathologies.…”