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

Abstract

The present report describes the effects of orthophosphate on the kinetic and physical properties of human glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14). 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 concentration of 50 mM did not alter the interaction coefficient for AMP or GMP. The 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. It is concluded from these studies that Pi acts as a substrate analogue for PP-ribose-P, and 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.