Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase

Abstract

A procedure is described for a 450-fold purification of 5-phosphoribosyl α-1-pyrophosphate (PRPP) synthetase from cells of Salmonella typhimurium LT-2. A sedimentation coefficient (s20, w) of 17.7 was estimated for the enzyme by sucrose density gradient centrifugation. Some general properties of the enzyme are described. PRPP synthetase has a specific and apparently absolute requirement for high levels of inorganic phosphate for PRPP synthesis. Saturation curves for phosphate are bimodal, which suggests multiple effects of the anion on the system. Phosphate appears to have a role in maintaining the structural integrity of the enzyme, since removal of phosphate by dialysis or dilution inactivates the enzyme. The enzyme requires a divalent cation such as Mg++ or Mn++ for activity. Kinetic studies indicate that the active substrate is the Mg-ATP complex, and that the divalent cation also activates the system in some other way, possibly by activating the enzyme. The PRPP synthetase reaction was demonstrated to be reversible. The equilibrium constant for the reaction in the direction of PRPP synthesis at pH 7.5 and 37° was determined to be 28.6, which corresponds to a standard free energy (ΔF‡) of −2.0 ± 0.5 kcal per mole for the reaction. The standard free energy of hydrolysis of PRPP to ribose 5-phosphate and inorganic pyrophosphate was estimated to be −7.0 ± 0.5 kcal per mole.