The activities of the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells are kinetically independent.
The bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells has very different kinetic properties from the larger NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase present in all mammalian cells. The NAD-dependent dehydrogenase is unique in that it requires formation of a magnesium.enzyme complex to allow addition of the first substrate, NAD+. It catalyzes an equilibrium ordered kinetic mechanism that has methylenetetrahydrofolate as the last reactant to add and NADH as the last product released. The NADP-dependent dehydrogenase has the same order of addition of substrates, but NADPH is released prior to methenyltetrahydrofolate. The dehydrogenase-cyclohydrolase activities of both enzymes channel methenyltetrahydropteroylglutamate intermediates with the same efficiency which is unaffected by the number of glutamyl residues in the methylenetetrahydrofolate substrate. However, the cyclohydrolase activity of the bifunctional protein is kinetically independent of its dehydrogenase activity, as supported by its lack of inhibition by NAD+, whereas NADP+ strongly inhibits that of the NADP-dependent enzyme. This difference is further demonstrated by the observation that conversion of formyltetrahydrofolate to methylenetetrahydrofolate in the presence of reduced pyridine nucleotide is catalyzed readily only by the bifunctional enzyme.
Rios-Orlandi
E M
EM
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
MacKenzie
R E
RE
D000619:Aminohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*
D009097:Multienzyme Complexes; C055342:methylene tetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; D008345:Manganese; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D008274:Magnesium