Studies on the polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver

• Published in: Biochemistry (Easton) 1984-04, Vol.23 (8), p.1796-1801
• Main Authors: Ross, Jonathan, Green, Jacalyn, Baugh, Charles M, MacKenzie, Robert E, Matthews, Rowena G
• Format: Article
• Language: English
• Subjects: 5,10-methylene tetrahydrofolate reductase (FADH2); Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Enzymes and enzyme inhibitors; Folic Acid - analogs & derivatives; Fundamental and applied biological sciences. Psychology; Kinetics; liver; Liver - enzymology; Methylenetetrahydrofolate Dehydrogenase (NADP) - isolation & purification; Methylenetetrahydrofolate Dehydrogenase (NADP) - metabolism; Oxidoreductases; Oxidoreductases - metabolism; pigs; poly(glutamic acid); Pteroylpolyglutamic Acids - metabolism; Pteroylpolyglutamic Acids - pharmacology; Structure-Activity Relationship; Substrate Specificity; Swine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00303a033

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.