Rat liver phosphoribosylpyrophosphate synthetase is activated by free Mg 2+ in a manner that overcomes its inhibition by nucleotides

Abstract

Phosphoribosylpyrophosphate synthetase is activated by Pi and free Mg 2+ as an essential activator and inhibited by nucleotides, especially ADP and GDP. The rat liver enzyme is a complex aggregate of two highly homologous catalytic subunits (PRS I and PRS II) and two associated proteins (PAP39 and PAP41). PRS I is more sensitive to inhibition by ADP and GDP than is PRS II. The native liver enzyme showed a weaker sensitivity to inhibition by nucleotides than expected from its composition. To further understand the regulation of the liver enzyme, kinetic studies of each subunit component and the liver enzyme regarding Mg 2+ activation and inhibition by ADP and GDP were carried out. Assay conditions were designed to keep free Mg 2+ at constant concentrations. (1) GDP, as MgGDP, did not affect the apparent K m values of PRS I for MgATP and ribose-5-phosphate but did dramatically increase the apparent K a value for free Mg 2+. (2) In contrast, ADP, as MgADP, increased the K m value for MgATP of PRS I as well as the K a value for free Mg 2+. (3) High concentrations of free Mg 2+ almost completely nullified the inhibitory effect of MgGDP and partly that of MgADP on PRS I. (4) At low free Mg 2+ concentrations within the physiological range, inhibition by the nucleotides is of physiological significance and conversely, variation in free Mg 2+ concentrations critically affects the enzyme activity in the presence of inhibitory nucleotides. (5) The response of PRS II and the native liver enzyme is similar to that of PRS I, while the effects of MgGDP and MgADP were smaller than that on PRS I. (6) We propose that MgGDP binds to a regulatory site of PRS I and PRS II and MgADP to the substrate MgATP site and also the regulatory site. The allosteric interaction of the regulatory site and the Mg 2+ binding site is also considered.