Probing the cellular role of the folylpolyglutamate chain: Studies of methylene tetrahydrofolate dehydrogenase and methylene tetrahydrofolate reductase.

Folic acid, an essential vitamin for mammals, is the carrier of activated one-carbon units in metabolism. While folic acid is transported in the blood as methyltetrahydropteroylmonoglutamate, it exists in mammalian cells with a poly-$\gamma$-glutamate tail. One function of this polyglutamate tail may be to regulate the flux of folates through the various cellular pathways. One experimental approach to determine whether the folylpolyglutamate tail has such a role in regulation is to compare the polyglutamate specificities of the folate-dependent enzymes from one source. This thesis examined the polyglutamate specificity of methylenetetrahydrofolate dehydrogenase of pig liver using steady-state kinetics and kinetic isotope effects. One of four methylenetetrahydrofolate-dependent enzymes in pig liver, methylenetetrahydrofolate dehydrogenase is the least sensitive to the folylpolyglutamate chain, and binds pentaglutamate inhibitors only three to seven fold tighter than monoglutamates. The kinetic parameters of methylenetetrahydrofolate dehydrogenase were measured and used in a calculation of the initial velocities of pig liver methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase, at physiological concentrations of substrates. These calculations suggested that the cellular concentration of methylenetetrahydrofolate may regulate the flux of methylenetetrahydrofolate into the pathways leading to nucleotide biosynthesis and methionine regeneration. High cellular levels of methylenetetrahydrofolate permit more one-carbon units to be directed towards nucleotide biosynthesis. This thesis also examined the potential role of methylenetetrahydrofolate reductase in the uptake of methyltetrahydropteroylmonoglutamate from the blood. Methylenetetrahydrofolate reductase and cobalamin-dependent methionine synthase are the only two enzymes known to react with methyltetrahydrofolate. It had been proposed that methylenetetrahydrofolate reductase might catalyze the oxidation of methyltetrahydropteroylmonoglutamate to methylenetetrahydropteroylmonoglutamate with concomitant reduction of an electron accepter such as menadione or methylenetetrahydropteroylhexaglutamate. Steady-state and rapid-reaction methods were used to show that in the presence of cellular concentrations of S-adenosylmethionine and NADPH, methylenetetrahydrofolate reductase could not play a role in the incorporation of methyltetrahydrofolate into cellular metabolism.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128151/2/8812897.pd