title: Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase.
abstract: An NAD(+)-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase has been purified to homogeneity from the yeast Saccharomyces cerevisiae. The purified enzyme exhibits a final specific activity of 5.4 units mg-1 and is represented by a single protein of apparent Mr = 33,000-38,000 as determined by sodium dodecyl sulfate gel electrophoresis. A native Mr = 64,000 was determined by gel filtration, suggesting a homodimer subunit structure. Cross-linking experiments with dimethyl suberimidate confirmed the dimeric structure. The enzyme is specific for NAD+ and is not dependent on Mg2+ for activity. The forward reaction initial velocity kinetics are consistent with a sequential reaction mechanism. With this model, Km values for NAD+ and (6R,S)-5,10-methylene-THF are 1.6 and 0.06 mM, respectively. In contrast to all other previously described eukaryotic 5,10-methylene-THF dehydrogenases, the purified enzyme is apparently monofunctional, with undetectable 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities. Subcellular fractionation of yeast indicates the enzyme is cytoplasmic, with no NAD(+)-dependent 5,10-methylene-THF dehydrogenase detectable in mitochondria. The activity was found in all yeast strains examined, at all stages of growth from the lag phase through the stationary phase.
authors.affiliation: Department of Chemistry, University of Texas, Austin 78712.
authors.forename: C K
authors.initials: CK
authors.lastname: Barlowe
authors.forename: D R
authors.initials: DR
authors.lastname: Appling
mesh_terms: D007700:Kinetics; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D009243:NAD; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP)