Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates

Journal Article · Tue Dec 13 04:00:00 EST 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00425a027· OSTI ID:6206706

Banerjee, R V; Shane, B; McGuire, J J; Coward, J K

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), Journal Name: Biochemistry; (United States) Vol. 27:25; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
ANTIMETABOLITES
AROMATICS
AZAARENES
BIOSYNTHESIS
CARBOXYLIC ACIDS
CATALYSIS
DRUGS
ENZYMES
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
FOLIC ACID
GLUTAMIC ACID
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPES
ISOTOPIC EXCHANGE
LABELLED COMPOUNDS
LIGASES
LIGHT NUCLEI
MAGNETIC RESONANCE
MASS SPECTROSCOPY
METHOTREXATE
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN 17
OXYGEN 18
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHORUS 31
PHOSPHORUS ISOTOPES
PTERIDINES
RESONANCE
SPECTROSCOPY
STABLE ISOTOPES
SUBSTRATES
SYNTHESIS
VITAMIN B GROUP
VITAMINS
WATER