title: Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates.
abstract: The transfer of 17O and/or 18O from (COOH-17O or -18O) enriched substrates to inorganic phosphate (Pi) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-18O-labeled folate, methotrexate, and dihydropteroate, in addition to [17O]-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. Pi was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for 17O and 18O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate 18O-enriched carboxyl group to Pi occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of Pi obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that 18O transfer had occurred.
authors.affiliation: Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: R V
authors.initials: RV
authors.lastname: Banerjee
authors.forename: B
authors.initials: B
authors.lastname: Shane
authors.forename: J J
authors.initials: JJ
authors.lastname: McGuire
authors.forename: J K
authors.initials: JK
authors.lastname: Coward
mesh_terms: D000818:Animals; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005492:Folic Acid / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008727:Methotrexate / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes; D010453:Peptide Synthases / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D011622:Pterins / Q000378:metabolism; D051381:Rats; D013552:Swine
chemical_list: D005971:Glutamates; D010755:Organophosphates; D010103:Oxygen Isotopes; D010710:Phosphates; D011622:Pterins; C000978:dihydropteroate; D018698:Glutamic Acid; D005492:Folic Acid; D010453:Peptide Synthases; C020041:dihydrofolate synthetase; C023103:folylpolyglutamate synthetase; D010100:Oxygen; D008727:Methotrexate; C003715:trimethyl phosphate