title: Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase.
abstract: A human mitochondrial isozyme of C1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C1-THF synthase. All C1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H4-PteGlu1, (6R,S)-H4-PteGlu3, and (6R,S)-H4-PteGlu5 substrates. The (6R,S)-H4-PteGlun Km dropped from greater than 500 microM for the monoglutamate to 15 microM and 3.6 microM for the tri- and pentaglutamates, respectively. The Km values for formate and ATP also are lowered when THF polyglutamates are used. The formate Km dropped 79-fold and the ATP Km dropped more than 5-fold when (6R,S)-H4-PteGlu5 was used as the substrate in place of (6R,S)-H4-PteGlu1.
authors.affiliation: Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A5300, Austin, TX 78712, USA.
authors.forename: Addie S
authors.initials: AS
authors.lastname: Walkup
authors.forename: Dean R
authors.initials: DR
authors.lastname: Appling
mesh_terms: D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D005574:Formate-Tetrahydrofolate Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005971:Glutamates; D007527:Isoenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase