Characterization and Chemical Properties of Phosphoribosylamine, an Unstable Intermediate in the de Novo Purine Biosynthetic Pathway

F. J. Schendel, Y. S. Cheng, J. D. Otvos, J. Stubbe, J. D. Otvos

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Abstract

Incubation of [l-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 [l-13C]ribose 5-phosphate with NH3. Extensive NMR studies (15N and1H-13C chemical shift correlation spectra) have allowed assignment of these resonances to β- and α-phosphoribosylamine. These studies represent the first spectral observations of this chemically reactive intermediate. The rate of interconversion of α- to β-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 NH3and 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 °C and pH 7.5. The pH-independent equilibrium constant for ribose 5-phosphate and NH3with 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 α- and β-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.

Original languageEnglish (US)
Pages (from-to)2614-2623
Number of pages10
JournalBiochemistry
Volume27
Issue number7
DOIs
StatePublished - Apr 1 1988

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