Regulation of the mammalian carbamoyl‐phosphate synthetase II by effectors and phosphorylation

We have measured the ‘core’ mammalian carbamoyl‐phosphate synthetase II (CPSII) activity, using NH 4 Cl as the nitrogen‐donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving K m (NH 3 ) values of 166 μM at high ATP concentrations and 26 μM at low ATP concentrations, while the K m (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the K m (NH 3 ) are lower than previously reported for CPS II, and closer to the values for the mitochondrial counterpart. The K m for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS II activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of actviity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5‐phosphoribosyl diphosphate ( P Rib PP ), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS II enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low‐affinity UTP‐stabilised state. Using our improved assay procedure, we observe a very large activation by P Rib PP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator P Rib PP is able to act on the phosphorylated enzyme.