Folylpolyglutamate synthetase: direct evidence for an acyl phosphate intermediate in the enzyme-catalyzed reaction

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5246973

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

The nature of the intermediate in the reaction catalyzed by folylpoly-..gamma..-glutamate synthetase (FPGS) has been investigated. Incubation of ..cap alpha..,..gamma..-(/sup 18/O)methotrexate with ATP, glutamate, and FPGS resulted in the formation of (/sup 18/O)phosphate, thus providing strong evidence for the formation of a ..gamma..-glutamyl phosphate during catalysis. The inorganic phosphate formed in the enzyme-catalyzed reaction was separated from other products and substrates by chromatography on DEAE-cellulose, then converted to the trimethyl ester, and analyzed by mass spectroscopy. Stoichiometric formation of (/sup 18/O)phosphate was observed in the case of the E. coli enzyme, isolated from a transformant containing the cloned FPGS-dihydrofolate synthetase (folC) gene. In addition, /sup 31/P-NMR analysis of the phosphate isolated from the reaction using E. coli FPGS showed the expected /sup 18/O-isotopic perturbations due to both singly bonded and doubly bonded P-/sup 18/O species. Similar experiments were carried out with FPGS isolated from hog liver. In this case, the small amounts of pure enzyme available precluded use of the NMR technique. However, mass spectral analysis of the derivatized phosphate product revealed the presence of (/sup 18/O)-trimethyl phosphate, thus indicating that the reaction catalyzed by the mammalian enzyme also proceeds via an acyl phosphate intermediate.

• Research Organization: Rensselaer Polytechnic Institute, Troy, NY
• OSTI ID: 5246973
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550600 -- Medicine
59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
ANIMALS
ANTIMETABOLITES
ATP
BACTERIA
BIOCHEMICAL REACTION KINETICS
BODY
CARBOXYLIC ACIDS
CATALYSIS
CHROMATOGRAPHY
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
DRUGS
ENZYMES
ESCHERICHIA COLI
EVEN-EVEN NUCLEI
GLANDS
GLUTAMIC ACID
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGASES
LIGHT NUCLEI
LIVER
MAMMALS
MASS SPECTROSCOPY
METHOTREXATE
MICROORGANISMS
NMR SPECTRA
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
OXYGEN 18
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHATES
PHOSPHORUS 31
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
SPECTRA
SPECTROSCOPY
STABLE ISOTOPES
SWINE
TRACER TECHNIQUES
VERTEBRATES