Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase

Phosphoribosylpyrophosphate (PRPP) synthetase from Salmonella typhimurium LT-2 catalyzes an exchange between 14C-AMP and ATP in the absence of added ribose 5-phosphate. The exchange reaction requires Mg++ ions and low concentrations of inorganic phosphate in addition to the substrates. High concentrations of inorganic phosphate (which stimulate over-all PRPP synthesis and inhibit the over-all reverse reaction) inhibit the exchange reaction. Low concentrations (0.2 mm) of ribose 5-phosphate stimulate the exchange reaction from 10- to 55-fold depending on the pH of the reaction mixture, but higher concentrations are strongly inhibitory. The Km values for the substrates are not substantially altered by ribose 5-phosphate stimulation. PRPP synthetase also catalyzes an exchange between 14C-ribose 5-phosphate and PRPP in the absence of added adenine nucleotides. The Mg++ and phosphate requirements for this exchange reaction are similar to those of the AMP-ATP exchange reaction. Ribose 5-phosphate-PRPP exchange is stimulated more than 200-fold by low concentrations (0.1 mm) of AMP, but is strongly inhibited by higher concentrations of AMP. AMP stimulation is not a consequence of changes in Km values for the substrates. The maximal rates of the unstimulated AMP-ATP exchange reaction and of the unstimulated ribose 5-phosphate-PRPP exchange reaction are slower than the maximal rate of the over-all reverse reaction of PRPP synthetase, but the rates of the stimulated exchanges are at least as fast as the over-all reactions of PRPP synthetase. Evidence is presented that the unstimulated exchange reactions can occur in the complete absence of the stimulating partner substrates. The results are interpreted as favoring a mechanism in which an enzyme-pyrophosphate intermediate is formed during the PRPP synthesis reaction, but in which this intermediate normally occurs in a ternary complex with AMP or ribose 5-phosphate (or both) noncovalently bound. The stimulation of exchange by AMP and ribose 5-phosphate is viewed as an example of substrate synergism (Bridger, W. A., Millen, W. A., and Boyer, P. D., Biochemistry, 7, 3608 (1968)).