Interactions of pig liver methylenetetrahydrofolate reductase with methylenetetrahydropteroylpolyglutamate substrates and with dihydropteroylpolyglutamate inhibitors
Dihydrofolate and dihydropteroylpolyglutamates inhibit pig liver methylenetetrahydrofolate reductase. In all cases the inhibition is linearly competitive with respect to methylenetetrahydrofolate. The Ki values decrease with each additional glutamyl residue from 1 to 6, from a value of 6.5 .mu.M for dihydrofolate to 0.013 .mu.M for dihydropteroylhexaglutamate. Dihydropteroylheptaglutamate has a Ki of 0.065 .mu.M. These data indicate a free energy of binding of .apprx. 0.75 kcal/mol for each of the 5 terminal glutamyl residues in dihydropteroylhexaglutamate. Methylenetetrahydropteroylpolyglutamates are substrates for the enzyme, and the increased free energy of binding is reflected in increased values for Vmax/Km with polyglutamate substrates. Vmax is increased 1.76-fold on going from the mono- to the diglutamate substrate; additional glutamyl residues lead to decreases in Km values for methylenetetrahydropteroylpolyglutamates. The in vivo activity of methylenetetrahydrofolate reductase may also be sensitive to fluctuations in the ratio of methylenetetrahydropteroylpolyglutamates to dihydropteroylpolyglutamates and this ratio may be important in determining the relative fluxes of methylenetetrahydropteroylpolyglutamates into the pathways leading to thymidylate biosynthesis and methionine regeneration.