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 [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.[1]References
- Studies on the polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver. Ross, J., Green, J., Baugh, C.M., MacKenzie, R.E., Matthews, R.G. Biochemistry (1984) [Pubmed]
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