title: Studies on the polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver.
abstract: 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 [Cohen, L., & MacKenzie, R. E. (1978) Biochim. Biophys. Acta 522, 311-317]. We have studied the inhibition of this activity by a series of pteroylglutamates containing one to seven glutamyl residues. Inhibitors with one to four glutamyl residues exhibit a kinetically determined KD of about 56 microM for binding at the folate site of the enzyme, while inhibitors with five to seven glutamyl residues exhibit a KD of about 16 microM. These results suggest that folylpolyglutamates are 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. We have also measured the kinetic parameters Vmax and the Km values for NADP+ and the folate substrate associated with catalysis using a series of methylenetetrahydropteroylpolyglutamate substrates. The variation in these parameters with the length of the polyglutamate tail is small.
mesh_terms: D000818:Animals; D005492:Folic Acid / Q000031:analogs & derivatives*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
chemical_list: D011624:Pteroylpolyglutamic Acids; D005492:Folic Acid; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP)