Interactions of Pig Liver Methylenetetrahydrofolate Reductase with Methylenetetrahydropteroylpolyglutamate Substrates and with Dihydropteroylpolyglutamate Inhibitors

Abstract

Dihydrofolate and dihydropteroylpolyglutamates inhibit pig liver methylenetetrahydrofolate reductase. In all cases the inhibition is linearly competitive with respect to methylenetetrahydrofolate. The K1 values decrease with each additional glutamyl residue from one to six, from a value of 6.5 ¼M for dihydrofolate to 0.013 ¼M for dihydropteroylhexaglutamate. Dihydropteroylheptaglutamate has a K1 of 0.065 ¼M These data indicate a free energy of binding of ~ 0.75 kcal/mol for each of the five 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. Kmax is increased 1.76-fold on going from the mono-to the diglutamate substrate; additional glutamyl residues lead to decreases in Am values for methylenetetrahydropteroylpolyglutamates. Our results suggest that the in vivo activity of methylenetetrahydrofolate reductase may also be sensitive to fluctuations in the ratio of methylenetetrahydropteroylpolyglutamates to dihydropteroylpolyglutamates and that this ratio may be important in determining the relative fluxes of methylenetetrahydropteroylpolyglutamates into the pathways leading to thymidylate biosynthesis and methionine regeneration. © 1980, American Chemical Society. All rights reserved.