Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates
The transfer of /sup 17/O and/or /sup 18/O from (COOH-/sup 17/O or -/sup 18/O) enriched substrates to inorganic phosphate (P/sub i/) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-/sup 18/O-labeled folate, methotrexate, and dihydropteroate, in addition to (/sup 17/O)-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. P/sub i/ was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for /sup 17/O and /sup 18/O 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 /sup 18/O-enriched carboxyl group to P/sub i/ 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 P/sub i/ 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 /sup 18/O transfer had occurred.
- Research Organization:
- Rensselaer Polytechnic Institute, Troy, NY (USA)
- OSTI ID:
- 6206706
- Journal Information:
- Biochemistry; (United States), Vol. 27:25
- Country of Publication:
- United States
- Language:
- English
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FOLIC ACID
BIOSYNTHESIS
LIGASES
NUCLEAR MAGNETIC RESONANCE
OXYGEN 17
ISOTOPIC EXCHANGE
OXYGEN 18
CATALYSIS
GLUTAMIC ACID
LABELLED COMPOUNDS
MASS SPECTROSCOPY
METHOTREXATE
PHOSPHORUS 31
SUBSTRATES
WATER
AMINO ACIDS
ANTIMETABOLITES
AROMATICS
AZAARENES
CARBOXYLIC ACIDS
DRUGS
ENZYMES
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHORUS ISOTOPES
PTERIDINES
RESONANCE
SPECTROSCOPY
STABLE ISOTOPES
SYNTHESIS
VITAMIN B GROUP
VITAMINS
550601* - Medicine- Unsealed Radionuclides in Diagnostics