Regulation of human amidophosphoribosyltransferase: Interaction of orthophosphate, PP-ribose-P, and purine ribonucleotides

Abstract

The effects of Pi on the kinetic and physical properties of human glutamine phosphoribosylpyrophosphate (PP-ribose-P) amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14 are reported. Enzyme activity was shown to be dependent on the concentration of Pi relative to that of PP-ribose-P and purine ribonucleotides. The Hill coefficient for PP-ribose-P decreased from 1.83 .+-. 0.08 to 1.11 .+-. 0.04 when Pi was increased from 0 to 50 mM. Twenty-five millimolar PPi, 50 mM SO4 and 100 mM KCl did not alter the Hill coefficient for PP-ribose-P. In 50 mM Pi, the purine ribonucleotides AMP and GMP increased the Hill coefficient for PP-ribose-P from 1.1 to 1.6 and 1.8, respectively. At Pi concentrations less than 6.25 mM, purine ribonucleotides did not enhance the PP-ribose-P cooperativity which was already demonstrable. Pi at a concentrations of 50 mM did not alter the interaction coefficient for AMP or GMP. The [sedimentation coefficient] S20,w was 6.2 in both 6.25 mM Pi and 50 mM Pi in the absence of purine ribonucleotides. When both PP-ribose-P and purine ribonucleotides were included in the sucrose-density gradients, Pi did not alter the relative distribution between the small and large forms of the enzyme. Pi probably acts as a substrate analogue for PP-ribose-P. This may explain the mechanism by which Pi modulates the response of amidophosphoribosyltransferase to alterations in the concentrations of PP-ribose-P and purine ribonucleotides.