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Direct evidence for an acyl phosphate intermediate in the folylpoly-. gamma. -glutamate synthetase and dihydrofolate synthetase-catalyzed reactions

Thesis/Dissertation ·
OSTI ID:6945327
The mechanism of the reactions catalyzed by two enzymes, namely dihydrofolate synthetase (DHFS) and folylpoly-..gamma..-glutamate synthetase (FPGS), has been investigated. The nature of the intermediate in each of the two reactions was monitored simultaneously in the multifunctional enzyme, FPGS/DHFS from E. coli. The latter was isolated from a transformant containing the cloned FPGS/DHFS gene. Incubation of (/sup 18/O)-H/sub 2/Pte and (/sup 17/O)-glutamate with ATP and the enzyme, resulted in the formation of (/sup 18/O)- and (/sup 17/O)-P/sub i/, thus providing strong evidence for the formation of an acyl phosphate species during catalysis of each reaction. The inorganic phosphate formed in the enzyme-catalyzed reaction was purified by chromatography on DEAE-cellulose, then converted to the trimethyl ester and analyzed by mass spectroscopy /sup 17/O NMR and /sup 31/P NMR. Stoichiometric formation of (/sup 17/O)- and (/sup 18/O)-Pi was observed. /sup 31/P NMR analysis showed the expected /sup 18/O-induced isotopic perturbations. The presence of (/sup 17/O)-trimethyl phosphate was revealed by /sup 17/O NMR. The mechanism of the FPGS-catalyzed reaction was also investigated with the antifolate (/sup 18/O)-methotrexate.
Research Organization:
Rensselaer Polytechnic Inst., Troy, NY (USA)
OSTI ID:
6945327
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTIMETABOLITES
ATP
BACTERIA
BIOCHEMICAL REACTION KINETICS
CATALYSIS
CHROMATOGRAPHY
DRUGS
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGASES
LIGHT NUCLEI
MASS SPECTROSCOPY
METHOTREXATE
MICROORGANISMS
NMR SPECTRA
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
OXYGEN 17
OXYGEN 18
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHATES
PHOSPHORUS 31
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
SPECTRA
SPECTROSCOPY
STABLE ISOTOPES
STOICHIOMETRY
TRACER TECHNIQUES