RAT-LIVER PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE IS ACTIVATED BY FREEMG2+ IN A MANNER THAT OVERCOMES ITS INHIBITION BY NUCLEOTIDES

Phosphoribosylpyrophosphate synthetase is activated by Pi and free Mg2 + as an essential activator and inhibited by nucleotides, especially A DP and GDP. The rat liver enzyme is a complex aggregate of two highly homologous catalytic subunits (PRS I and PRS II) and two associated pr oteins (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 sensi tivity to inhibition by nucleotides than expected from its composition . To further understand the regulation of the liver enzyme, kinetic st udies of each subunit component and the liver enzyme regarding Mg2+ ac tivation and inhibition by ADP and GDP were carried out. Assay conditi ons were designed to keep free Mg2+ at constant concentrations. (1) GD P, 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 Mg2+. (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 Mg2+. (3) High concentrations of free Mg2+ almost completely nullified the inhi bitory effect of MgGDP and partly that of MgADP on PRS I. (4) At low f ree Mg2+ concentrations within the physiological range, inhibition by the nucleotides is of physiological significance and conversely, varia tion in free Mg2+ concentrations critically affects the enzyme activit y in the presence of inhibitory nucleotides. (5) The response of PRS I I and the native liver enzyme is similar to that of PRS I, while the e ffects of MgGDP and MgADP were smaller than that on PRS I. (6) We prop ose that MgGDP binds to a regulatory site of PRS I and PRS II and MgAD P to the substrate MgATP site and also the regulatory site. The allost eric interaction of the regulatory site and the Mg2+ binding site is a lso considered. (C) 1998 Elsevier Science B.V. All rights reserved.