Evidence against Formation of a Pyrophosphoryl Enzyme Intermediate in the Phosphoribosylpyrophosphate Synthetase Reaction

Highly purified phosphoribosylpyrophosphate (PRPP) synthetase from Salmonella typhimurium reacts with [γ-32P]ATP, but binds not more than 0.001 mole of phosphate per mole of enzyme subunit under conditions optimal for the PRPP synthetase reaction. Use of [γ-32P]-, [β, γ-32P]-, [β-32P]-, and [3H]ATP showed that the bound radioactivity probably arose from very tight binding of intact ATP and binding of the γ-phosphate of ATP, possibly to a trace contaminant in the enzyme preparation. A series of analogues of ribose-5-P was investigated as substrates and inhibitors of PRPP synthetase. One of these, 1,4-anhydro-dl-ribitol-5-P, was devoid of activity as a substrate and was a competitive inhibitor with respect to ribose-5-P. This analogue effectively inhibited [14C]AMP-ATP exchange catalyzed by PRPP synthetase, which exchange had been previously shown to be stimulated by ribose-5-P. This result indicates that ribose-5-P probably stimulates the AMP-ATP exchange reaction by acting as an intermediate pyrophosphoryl group carrier rather than by inducing changes in the rate of an already existing exchange reaction, i.e. acting as a substrate synergist. The failure to isolate a pyrophosphoryl enzyme and the demonstration of the inability of PRPP synthetase to catalyze AMP-ATP exchange in the absence of ribose-5-P controvert previously published evidence in favor of participation of a pyrophosphoryl enzyme intermediate in the PRPP synthetase reaction. Simple chemical syntheses of methyl (α,β)-d-ribofuranoside-5-P and 1,4-anhydro-dl-ribitol-5-P are presented.