Characterization and chemical properties of phosphoribosylamine, an unstable intermediate in the de novo purine biosynthetic pathway

Incubation of 1-13C]-5-phosphoribosyl pyrophosphate (1-13C]PRPP) and glutamine with PRPP amidotransferase results in rapid production and disappearance of two new resonances at 89.3 and 85.9 ppm. These resonances coincide with two of the products produced upon incubation of 1-13C]ribose 5-phosphate with NH3. Extensive NMR studies (15N and 1H-13C chemical shift correlation spectra) have allowed assignment of these resonances to beta- and alpha-phosphoribosylamine. These studies represent the first spectral observations of this chemically reactive intermediate. The rate of interconversion of alpha- to beta-phosphoribosylamine as a function of pH has been determined by saturation and inversion-transfer NMR methods. The rate of formation of 5-phosphoribosylamine (PRA) from ribose 5-phosphate and NH3 and its rate of decomposition as a function of pH have been determined with a glycinamide ribonucleotide synthetase trapping system fashioned after earlier studies of Nierlich and Magasanik Nierlich, D. P., & Magasanik, B. (1965) J. Biol. Chem. 240, 366]. Phosphoribosylamine has a t1/2 = 38 s at 37 degrees C and pH 7.5. The pH-independent equilibrium constant for ribose 5-phosphate and NH3 with phosphoribosylamine has been established, 2.5 M-1, by use of these rate constants as well as by NMR methods. This equilibrium constant and the rates of nonenzymatic interconversion of alpha- and beta-PRA provide essential background for studying the mechanism of glycinamide ribonucleotide synthetase and investigating the possibility of channeling phosphoribosylamine between this enzyme and the first enzyme in the purine pathway.