Guanosine monophosphate synthetase from ehrlich ascites cells. Multiple inhibition by pyrophosphate and nucleosides

Abstract

GMP synthetase (xanthosine-5′-phosphate: ammonia ligase (AMP-forming), EC 6.3.4.1) from Ehrlich ascites cells was found to be subject to multiple inhibition by its reaction product, PP i, and some analogs of adenosine. PP i and the nucleoside (N) inhibitors were also capable of individually inhibiting this enzyme. Under no conditions did the inhibition appear to be irreversible or “pseudoinactivating” in nature. The individual inhibition by PP i was competitive with respect to ATP ( K 1 = 0.42 mM ). Conversely, in the absence of PP i, the binding of N was noncompetitive with ATP, but shifted to a competitive pattern when PP i was present. Furthermore, with the inhibitors in concert, there was an apparent lowering of the K I values for both inhibitors. This data was consistent with either PP i functioning to tighten the binding of N at a noncatalytic site (positive cooperativity) or with PP i actually opening a second binding site for N in addition to the non-catalytic site. Although this study did not distinguish which of these events was occurring, it did reveal that the intensity of the effect of PP i appeared to be constant. That is, for various N inhibitors with a range of independently determined K I values from 26 to 1650 μM, the ratio of their K I values determined in the absence of PP i to the values determined in the presence of PP i was always 38 ± 1.