STUDIES ON THE POLYGLUTAMATE SPECIFICITY OF METHYLENETETRAHYDROFOLATE DEHYDROGENASE FROM PIG-LIVER

Methylenetetrahydrofolate dehydrogenase, which is one of the activities of a trifunctional folate-dependent enzyme isolated from pig liver, displays an ordered bi-bi kinetic mechanism when methylenetetrahydropteroylmonoglutamate is used as the folate substrate. The inhibition of this activity by a series of pteroylglutamates containing 1-7 glutamyl residues was studied. Inhibitors with 1-4 glutamyl residues exhibit a kinetically determined Kd of .apprx. 56 .mu.M for binding at the folate site of the enzyme, while inhibitors with 5-7 glutamyl residues exhibit a Kd of .apprx. 6 .mu.M. Folypolyglutamates are probably bound to the trifunctional enzyme relatively weakly, with the major interaction involving the fifth glutamyl residue of the polyglutamate tail. A free energy decrease of about 0.74 kcal (3.1 kJ) is associated with this interaction. The possibility of a swinging arm mechanism for the trifunctional enzyme is discussed. The kinetic parameters Vmax and the Km values for NADP and the folate substrate associated with catalysis were also measured using a series of methylenetetrahydropteroylpolyglutamate substrates. The variation in these parameters with the length of the polyglutamate tail is small.