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

The mechanism of the reactions catalyzed by two enzymes, namely dihydrofolate synthetase (DHFS) and folylpoly-γ-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 [18O]-H2Pte and [17O]-glutamate with ATP and the enzyme, resulted in the formation of [18O]- and [17O]-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 17O NMR and 31P NMR. Stoichiometric formation of [17O]- and [18O]-Pi was observed. 31P NMR analysis showed the expected 18O-induced isotopic perturbations. The presence of [17O]-trimethyl phosphate was revealed by 17O NMR. The mechanism of the FPGS-catalyzed reaction was also investigated with the antifolate [18O]-methotrexate