Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase: III. KINETIC STUDIES OF THE REACTION MECHANISM

Abstract

Abstract An analysis of the kinetic mechanism of phosphoribosylpyrophosphate (PRPP) synthetase has been carried out with highly purified preparations of the enzyme from Salmonella typhimurium. The enzyme requires both MgATP2- as a substrate and free Mg2+ ion as an essential activator. This conclusion is supported by kinetic analysis and by studies of partial inhibition of PRPP synthetase by low concentrations of Ca2+ ions in the presence of excess MgCl2. The kinetics of Mg2+ and MgATP2- interaction with PRPP synthetase in the presence of saturating concentrations of ribose 5-phosphate is consistent with random, rapid equilibrium binding of Mg2+ and MgATP2- prior to rate-limiting breakdown of the central complex to yield products. The analysis of the kinetics was greatly facilitated by a computer program which calculated free Mg2+ concentration and the concentration of three Mg2+ complexes in a system in which Mg2+ is in equilibrium with three ligands. The results of initial velocity studies of the PRPP synthesis (forward) and ATP synthesis (reverse) reactions and product inhibition studies are consistent with an Ordered Bi Bi mechanism in which MgATP2- binds to the enzyme first and PRPP dissociates last. A previous study of partial exchange reactions catalyzed by PRPP synthetase (Switzer, R. L., J. Biol. Chem., 245, 483 (1970)) suggested an enzyme-pyrophosphate intermediate in the reaction. However, the characteristics of the over-all exchange reactions catalyzed by the enzyme indicated that such a derivative, if it participates in the over-all reaction, is not kinetically significant as a free intermediate. The present studies confirm this conclusion.